THRO>IB0313 Printed

RESEJ.RCH in the United

SUBCELLULAR

S. Lopaciuk",

Vol. States

8, pp. Ajj-465, Pergamon Press,

DISTRIBUTION OF FIBRINOGEN AND FACTOR XIII IN HUMAN BLOOD PLATELETS

K.M. Lovette,

(Received

19;b Inc.

Jan McDonaght, H.Y.K. ChuangS, Department of Pathology University of North Carolina School of Medicine Chapel Hill, N. C. 27514

11.2.1976.

Accepted

by

Editor

M.I.

and R.P. McDonagh

Barnhart)

ABSTRACT Fresh human platelets were disrupted by nitrogen decompression, and homogenates were fractionated by centrifugation in a linear gradient of sodium diatrizoate (18-33%). Five distinct fractions were obElectron microscopy showed that the membrane fraction was tained. free of contaminating subcellular organelles. The granule fraction contained primarily intact granules, with some mitochondria and some A portion of the granules was also distributed in swollen granules. the fractions above and below the rimary granule fraction. Total protein was found to be 1.95 mg/lO !J platelets, of which 0.18 mg was identified as fibrinogen. The ratio of platelet to plasma fibrinogen in whole blood was determined to be 1:30. Total factor XIII activity was 69 umole dansylcadaverine incorporation/109 platelets, and factor XIII activity was found to be equally distributed between 70% of the platelet fibrinogen was found in plasma and platelets. association with granules, with the remainder appearing primarily in the soluble fraction. Platelet factor XIII was localized in the Platelet cytoplasm, with 94% appearing in the soluble fraction. fibrinogen was released during the platelet release reaction induced by collagen, whereas factor XIII was not.

Presented in part at the Federation of American Societies for Experimental Biology, April, 1975 (Fed. Proc. 34: 344). *Performed while on leave from the Institute of Hematology, Chocimska 5,00-957 Warsaw, Poland. tTo whom correspondence should be addressed. Memorial Hospital, Brown University, Pawtucket, Rhode 3Present address: Island 02860.

454

SUB-PLATELET

F-I

23 F-XIII

INTRODUCTION Blood platelets play an important role in coagulation; and there is considerable interest in their characterization, both morphologically and biochemically, especially with regard to understanding how the subcellular constituents contribute to platelet function. Platelets contain both fibrinogen (l-3) and factor XIII (4,5). Estimates of the concentration of fibrinogen in human platlets range from 50 to 180 *g/log platelets (6,7). Platelet fibrinogen is similar to plasma fibrinogen, but it is not certain if the two proteins, isolated from different sources, are identical (8-10). Several investigators have concluded that platelet fibrinogen is divided into two pools, with about 25 to 33 percent associated with granules and the remainder in the soluble fraction (11-13). Platelet factor XIII is enzymatically similar to plasma factor XIII but structurally different. Platelet factor XIII consists of a single, dimerized subunit, ~2, while plasma factor XIII has the a subunit together with an inactive b_ subunit and circulates as a complex (14,15). By means of a clot solubility assay platelet factor XIII has been reported to be in the soluble fraction of platelet homogenates (12). Disruption of platelet membranes and fractionation of the intracellular constituents present several problems in studying the subcellular distribution of platelet components. It is difficult to achieve complete disruption of platelets without excessive destruction of intracellular organelles. Further alteration of platelet organelles may result from osmotic stress in high density sucrose solutions. In the present studies human platelets were disrupted by nitrogen decompression and fractionated by density gradient centrifugation in sodium diatrizoate. These methods were used for studies on the subcellular localization of platelet fibrinogen and factor XIII. In corollary studies the release of these two proteins by collagen during the platelet release reaction was also characterized.

METHODS Subcellular fractionation. Human blood was collected from fasting, normal donors and anticoagulated with acid-citrate-dextrose (USP Formula A). All centrifugations for preparation of platelets were carried out at 20" in polycarbonate tubes in a Sorvall RC-3 centrifuge with swinging bucket rotor. Blood was centrifuged at 320 g for 15 min, and PRP' was removed and mixed with 2% EDTA in 0.7% NaCl, pH-7.4 (2O:l). PRP was recentrifuged at 320 g for 4 min to remove the remaining red cell contaminants. Platelets weresedimented by centrifugation at 1,800 g for 30 min and then resuspended in washing solution to l/3 volume of PRP.-The washing solution contained 0.14 M NaCl, 6 mM EDTA, 0.02 M Tris, pH 7.4. Platelets were sedimented by centrifugation at 320 g for 4 min, followed by 1,800 g for 8 min. Washing was repeated 3 times. Washed platelets were resuspended in 0.1 M KCl, 1 mM EDTA, 0.02 M Tris, pH 7.4, to a volume of 15 ml, and Trasylol R (FBA Pharmaceuticals) was added to a concentration of 50 KIU/ml. Platelet counts were determined by phase contrast microscopy and ranged from 2.2 to

=Abbreviations used in this paper: PRP, platelet-rich plasma; EDTA, disodium ethylenediamine tetraacetate; Tris, Tris(hydroxymethyl)amino-methane-HCl.

VOl.8,N0.0

ST-B-PLATELET F-I Zc F-XIII

3.0x10~/ml,with negligible erythrocyte contamination. Approximately 4 hr elapsed from the time blood was collected until the beginning of homogenization. Platelets were disrupted by explosive decompression with a procedure similar to that of Broekman -et al (16). The washed platelet suspension was equilibratedat 4" and then homogenized by nitrogen decompression in a Parr cell disruption bomb. Platelets were equilibrated with nitrogen at 1,500 lb/in2 (100 atm) for 20 min before explosive decompression. Linear gradients of sodium diatrizoate (sodium 3,5-diacetamido-2,4,6triiodobenzoate, Winthrop Laboratories), l&33%, containing 2 mM EDTA, were prepared. Osmolarity of the gradient was determined by the freezing point depression method with isotonic NaCl (275 mosmolar) as reference. For 18% sodium diatrizoate the osmolarity was 514 mosmolar, and at 33% sodium diatrizoate it was 960 mosmolar. As a comparison 30% sucrose was found to be 1025 mosmolar and 60% sucrose, 2066 mosmolar. Two ml of whole platelet homogenate were layered onto 10 ml of gradient and centrifuged at 4" at 193,000 hax for 3-3.5 hr in a Beckman L5-65 ultracentrifuge with SW36 rotor. All gradients displayed five distinct zones which were collected.separately. Each fraction was diluted with the resuspension buffer and centrifuged at 4" at 145,000 gmax for 30 min in a Type 40 rotor in order to separate the supernatant and particulate components. Particulate matter was washed and resuspended in a small volume of the same buffer. Electron microscopy. Aliquots of each fraction were fixed in 2.25% glutaraldehyde-0.1 M phosphate buffer for one hr and then centrifuged at 4" for 30 min at 145,000 gmax. The resulting small pellets were post-fixed in 0~04, dehydrated in ethanol, and imbedded in Epon. Ultrathin sections were stained with uranyl acetate and examined in the electron microscope. Total protein, fibrinogen concentration, and factor XIII activity of each subcellular fraction were determined immediately after density ultracentrifugation was completed. Assays for lactic dehydrogenase and B-Nacetyl-glucosaminidase were either performed immediately, or samples were rapidly frozen and stored overnight at -80". Protein determination. Total protein was assayed by a modified Lowry procedure (17) with crystalline bovine serum albumin as standard. Samples of homogenate and subcellular fractions were mixed with equal volumes of 1% sodium deoxycholate in 0.2 M NaOH and incubated at room temperature for 2-4 hr to ensure complete solubilization of structural proteins. Fibrinogen. Platelet fibrinogen was assayed as staphylococcal clumping reactive material (18). Staphylococcal clumping factor was obtained from Sigma Chemical Co. Fibrinogen standard was human plasma Fraction I-4 which was 98% clottable. The sensitivity of the assay was 1.25 ug fibrinogen/ml. Factor XIII. Platelet factor XIII activity was determined by the fluorescent amine incorporation method of Lorand -et al (19). Dansylcadaverine (N-(5-aminopentyl)-5-dimethylamino-l-naphthalenesulfonamide) was obtained from Sigma Chemical Co. Enzyme markers.

Lactic dehydrogenase (E.C.1.1.1.27) was assayed as

456

SUB-PLATELET

F-I

& F-XIII

described by Kornberg (20). 3-N-acetylglucosaminidase (E.C.3.2.1.30) was measured according to the method of Bosmann (21). Platelet release reaction. Blood was collected from normal, fasting, human donors who had not taken aspirin for 14 days, with 3.8% sodium citrate as anticoagulant. PRP was prepared by centrifugation at 320 g for 15 min. ['4C]serotonin (tiersham-Searle) was added to PRP to a final concentration of 1~10~~ M and incubated at 37' for 20 min. Platelet uptake of [14C)serotonin was greater than 95%. EDTA was added to the PRP (final concentration: 0.1X>, and platelets were sedimented by centrifugation at 1800 g for 20 min. Platelets were resuspended in l/5 volume of supernatant plasma. Platelets were separated from plasma by a minor modification of the method of Tangen -et al (22) using Sepharose 4B with modified Tyrode's solution (free of calcium and magnesium) as the elution buffer. Collagen was prepared from human cadaver Achilles tendon (23). Release studies were performed by incubating 0.9 ml platelet suspension (0.50.8x10g/m1) with 0.1 ml collagen. Controls contained saline in place of collagen. Samples were stirred magnetically for 5 min at 37' and then centrifuged at 8,000 g for 1 min. Aliquots of the supernatants were counted in a Nuclear Chicago liquid scintillation counter to determine serotonin release. Fibrinogen concentration and factor XIII activity in the supernatants were measured as described above.

RESULTS Density gradient fractionation of platelet homogenate. Platelet homogenates were separated into constituent elements by ultracentrifugation in a sodium diatrizoate gradient. Five distinct zones were observed and were collected as: (A) soluble fraction; (B) membrane fraction, which appeared to split into two zones; (C) intermediate fraction; (D) granule fraction; (E) bottom fraction. Each fraction containing particulate matter was subdivided into supernatant and particulate fractions. The border between the membrane and intermediate fractions was well defined, but there was no sharp interface between the intermediate and granule fractions or between the granules and the fraction at the bottom of the tube. From the gradient separation it appeared that some granules were also distributed in the intermediate and bottom fractions. This was confirmed by electron microscopy. Electron microscopy. Examination of whole platelet homogenates by transmission electron microscopy showed that platelet organelles were well preserved while the degree of disruption of platelet membranes by nitrogen decompression was high. Micrographs of the membrane fraction (Fig. 1) showed that membrane fragments appeared mainly as vesicles of different sizes and shapes, which contained varying amounts of amorphous material. This fraction appeared homogeneous and was largely uncontaminated by subcellular organelles. Electron micrographs of the intermediate fraction showed that it homogeneous and contained many membrane fragments mixed with granules mitochondria. Granules appeared to be dominant. Fraction D (Fig. 2) contained mainly granules, some mitochondria, and only a small amount membrane vesicles. The granules looked well preserved. &ny had the round, dense appearance of a-granules. Some had areas of eccentric

was and of typica1,

FIG. 1 Electron membrane

micrograp:h of platelet fraction. 23,000x.

FIG. 2 Electron micrograph of platelet granule fraction. 23,000x.

osmophilla, and some were swollen. xorphologic characterization of the small amount of sediment at the bottom of the gradient showed that this fraction contained a mixture of granules, swollen granules, mitochondria, platelet fragments with glycogen deposits, and debris. Total protein, fibrinogen, and factor XIII content of human platelets. 'Whole platelet homogenates were assayed for protein content, fibrinogen

rr%,_PL_Q-ELET --I

F-T & F-XTTT

concentration, and factor XIII activity (Table I). Homogenates were prepared from suspensions containing 2.2-3.0~10 washed platelets per ml. The average plasma fibrinogen concentration is 2.8 mg/ml plasma (24), and for plasma factor XIIIthe value is about 26 smole incorporation/ml plasma (25,26). Thus, th2 ratio of plasma fibrinogen to platelet fibrinogen is approximately 3O:l. In contrast the total factor XIII activity of blood is approximately evenly divided between the plasma and platelet components.

TABLE I Total Protein, Fibrinogen, and Factor XIII content in Platelet Homogenates Platelet count x log/m1

Protein mg/103 platelets

Fibrinogen mg/lOq platelets

2.8'0.3

1.95*0.26

0.18'0.03

(2.2-3-O)

(1.72-2.34)

(0.14-0.20)

Values are given as mean t SD.

Factor XIII nmole incorp./ lo3 platelets 69.4'13.1 (57.8-90.4)

Range is in parenthesis.

n = 5.

Subcellular distribution of platelet fibrinogen and factor XIII. Each fraction of platelet homogenate obtained after density gradient ultracentrifugation was assayed for total protein, Eibrinogen, and factor XIII. Lactic dehydrogenase was used as a marker for the soluble component and B-N-acetylglucosaminidase for the granules. Control experiments showed that sodium diatrizoate, at concentrations which appeared in the test samples, did not affect these determinations. Lactic dehydrogenase was found only in the soluble phase, with a recovery of 90.2%'5.3% (SD). The distribution of the other components in the subcellular fractions is given in Table II and Fig. For total protein, B-N-acetylglucosaminidase, and factor XIII, recoveries 3. ranged around 100%. For fibrinogen the recovery was 75% of that present in the homogenate. Approximately 69% of platelet fibrinogen was distributed in fractions C, D, and E, with the highest amount (300 pg/mg protein) in the particulate component of the granule fraction (D). Since all three of these fractions contained granules by electron microscopy, it appears that fibrinogen in each of these fractions was associated with granules. The distribution of platelet fibrinogen was similar to that of B-N-acetylglucosaminidase. 57% of the recovered platelet fibrinogen was located in the granule fraction CD). The distribution of fibrinogen differed from that of B-N-acetylglucosaminidase in that very little fibrinogen could be detected in the supernatants of the granule-containing fractions while significant E-N-acetylglucosaminidase was found in these supernatants. The distribution of 3-N-acetylglucosaminidase in the membrane fraction was significantly higher than that of fibrinogen. The data in Table II clearly show that platelet factor XIII, like lactic dehydrogenase, is a cytoplasmic component of platelets. 93.5% of the recovered platelet factor XIII activity was distributed in the soluble fraction, with 4% in the membrane fraction and only trace activity in the other fractions.

0.12+0.04

0.14$0.10

0.07$0.02

0.19-$.08

0.19$0.08

0.14+0.06

0.03tC - P02

B

C supernate

C particulate

D supemate

D particulate

E supernate

E particulate

53.2216.9

none detected

300.3i9.3

trace

131.2k5.5

trace

26.528.2

27.6t5.7

CLg/mg protein

Fibrinogen

3.021.2

trace

trace

trace

trace

trace

trace

14.8+4.2

63.7514.0

umole incorp./ mg protein*

none detected

57.2211.7

trace

8.4i3.7

trace

5.022.3

24.327.6

percent"

Factor XIII

.+Factor XIII activity is expressed as pmole dansylcadaverine incorporation/30 min/mg protein.

,kResults are expressed as per cent of the sum of recovered product.

2.4s.8

6.9k1.9

10.252.1

9.823.0

4.522.0

6.723.3

7.9i2.1

51.6+6.5

per cent??

All values are given as mean 2 SD. n = 5.

1.09$.10

mg/lOg platelets

Protein

A

Fraction

Distribution of Protein, Fibrinogen, and Factor XIII in Platelet Subcellular Fractions

TABLE II

trace

trace

trace

trace

trace

trace

4.1kl.O

93.5i2.4

per cent

460

SUB-PLATELET

F-I & F-XIII

IO0

90

80 BNACETYLGLUCOSAMINIDASE FIBRINOGEN

70

p%J FACTOR XIII

5 i= 2

60

cr $ o

50

g

40

z a

30 20

IO

0 ISOLUBLE

A

Supernate MEMBRANE

B

Particulate

INTERMEDIATE

C

Supernote

Particulate

GRANULE

Soluble Particulate BOTTOM

E FIG. 3

Distribution of S-N-acetylglucosaminidase, the fractions obtained by density gradient homogenates.

fibrinogen, and factor XIII in separation of human platelet

Release reaction induced by collagen. Gel filtered platelets were used for studies on the platelet release reaction (Table III). When platelets were exposed to collagen for 5 min, approximately 73% of [14C]serotonin was released into the ambient fluid. Similarly, 63% of platelet fibrinogen was released into the supernatant. In contrast only trace amounts of platelet factor XIII appeared in the supernatant under these experimental conditions.

SUB-PLATELET

F-I

461

d F-XIII

TABLE III Collagen-induced Release from Gel-filtered Platelets

Per cent released

Component tested 1

2

3

4

[l+C]serotonin

85.7

57.3

86.1

61.5

Fibrinogen

71.4

42.8

71.4

66.6

1.8

0.1

Factor XIII

0

0

For each component tested, % release = (amount in test supernatant 5 min after aggregation with collagen) - (amount in control supernatant)/(total amount in homogenate) - (amount in control supernatant). Results are given for 4 individual experiments.

DISCUSSION The physiologic functions of plasma fibrinogen and factor XIII are well defined, namely, fibrin formation and crosslinking. Fibrin is the primary substrate of activated factor XIII. The biological significance of fibrinogen and factor XIII which are compartmentalized in platelets is less clear, although it may be that one or both are involved in strengthening platelet aggregates. In order to characterize further platelet fibrinogen and platelet factor XIII, parallel studies were undertaken to determine the subcellular distribution of these proteins. Platelet membranes were disrupted by explosive decompression, and separation of the subcellular components was obtained by ultracentrifugation in a linear gradient of sodium diatrizoate. The primary advantages of this gradient are a relatively low osmolarity and viscosity, especially as compared to sucrose gradients, which result in less damage and better preservation of subcellular organelles. Nitrogen decompression produced a high yield of membrane disruption, and only a few intact platelets were observed by electron microscopy. Gradient separation on sodium diatrizoate produced a uniform membrane fraction, uncontaminated by granules or mitochondria (Fig. 1). The granule fraction (Fig. 2) contained a high proportion of intact granules, together with mitochondria and some damaged, swollen granules. Some granules, both intact and damaged, were also distributed in the intermediate fraction and the fraction below the granule layer. The gradient fractionation was highly reproducible. The concentration of platelet fibrinogen, as determined by the staphylococcal clumping procedure, was found to be 0.18'0.03 mg/lOy platelets. These results agree well with those obtained with immunochemical procedures (627). Platelet fibrinogen represents 9 per cent of total platelet protein (Table I). In other studies on platelet fibrinogen it has been reported that platelet fibrinogen is distributed in two compartments with about 6675% located in the soluble fraction and the remaining 25-33% associated with granules (11-13). It has also been reported that only the granule-associated

462

SUE-PLATELET

F-I

& F-XIII

Vo1.8,No.4

fibrinogen has physical and chemical characteristics which differ from plasma fibrinogen (27). Recently Broekman -et al (28) have also reported that platelet fibrinogen was associated with electron dense granules and platelet factor XIII was found in the supernatant, but they did not report quantitative values for platelet fibrinogen and factor XIII. In the present investigation approximately 70% of platelet fibrinogen was associated with granule containing fractions, with 57% appearing in the granule particulate fraction (Table II, Fig. 3). Only 5% of platelet fibrinogen was associated with platelet membranes. The higher content of granule fibrinogen found in these studies is probably due to the methods used for disruption and fractionation. These procedures resulted in better preservation of intact granules as shown by the distribution of the lysosomal marker, B-N-acetylglucosaminidase. In previous studies 50% or more of the lysosomal marker was found in the soluble fraction (11-13). In the present experiments 14% of .B-N-acetylglucosaminidasewas found in the soluble fraction, 19% in the membrane fraction, and the remaining 2/3 was found in the granule-containing fractions. Because of the high content of granule fibrinogen found in these experiments, it is concluded that platelet fibrinogen is mainly an intracellular, granule component. Fibrinogen appearing in the soluble and membrane fractions, representing less than l/3 of the total platelet fibrinogen, may have originated from granules which were disrupted during homogenization and fractionation. Some of this component might also be extracellular fibrinogen which was strongly adsorbed to the plasma membranes. The distribution of platelet fibrinogen was not fully correlated with that of B-N-acetylglucosaminidase. From these experiments it was not possible to determine which granules function as storage sites for fibrinogen. Granules containing the acid hydrolytic enzymes might be more labile structures than those containing fibrinogen, which would explain the difference in the subcellular distribution of 8-N-acetylglucosaminidase and fibrinogen. With regard to the subcellular distribution of platelet factor XIII, these experiments are clear. By quantitative determination 94% of platelet factor XIII was distributed in the soluble, cytoplasmic component (Table II, Fig. 3). The platelet content of factor XIII is high and accounts for approximately 50% of the total factor XIII activity in blood. Since it has also been shown that platelet factor XIII is present in megakaryocytes and that plasma factor XIII does not enter platelets -in vivo (29,30), it appears that platelet factor XIII is synthesized in megakaryocytes and becomes a cytoplasmic component of platelets as they are formed and released from the parent cells. In combination with studies on the subcellular distribution of platelet fibrinogen and platelet factor XIII, these proteins were also studied during the platelet release reaction induced by collagen. In previous studies it has been shown that platelet fibrinogen is released by thrombin (31-33). In the present studies platelet fibrinogen was also released by collagen (Table III). In the same test system only trace amounts of released platelet factor XIII could be detected by quantitative assay. The observations.agree with others in which a semi-quantitative, clot solubility assay was used to measure the release of platelet factor XIII (34). The results obtained for the release of platelet fibrinogen and platelet factor XIII by collagen are in agreement with the subcellular distribution studies and can be explained by the different subcellular localization of these two proteins.

Vol.P,To.4

SLq-PLATELET

F-I

&

F-XIII

The functions of platelet fibrinogen and platelet factor XIII remain to It has been suggested that fibrinogen, released from platelets be clarified. by thrombin, acts as a cofactor in subsequent aggregation (35). Also, as platelets aggregate they provide a catalytic surface for the plasma clotting It appears that intracellular fibrinogen, when released from system (36). aggregating platelets, may help to form the first strands of fibrin net. The biological function of platelet factor XIII requires further investigaIt is unclear whether or not the failure of factor XIII to be released tion. It is also in the platelet release reaction has any significance -in vitro. possible that, in addition to any function it may have in the initial stages of thrombus formation, platelet factor XIII may function as the source of the r?_subunit of plasma factor XIII.

ACKNOWLEDGEMENT We thank Dr. F. G. Dalldorf and H. L. Livingston, Department of Pathology, University of North Carolina School of Medicine, for preparing the electron micrographs.

REFERENCES 1. WARE, A. G., FAHEY, J. L., and SEEGERS, W. H. Platelet extracts, fibrin formation and interaction of purified prothrombin and thromboplastin. Amer. .I. Physiol. 154, 140, 1948. 2. GOKCEN, M. and YUNIS, E. Nature 200, 590, 1963.

Fibrinogen

3. CASTALDI, P. A. and CAEN, J. 579, 1965. 4. BULUK, K. An unknown 10, 191, 1955. 5. LUSCHER, Schweiz.

function

Platelet

as a part of platelet

fibrinogen.

of blood platelets.

E. F. Ein fibrinstabilisierender Med. Wochschr. 87, 1220, 1957.

structure.

.I. Clin. Path.

Polski Tygod

18,

Lekar

Faktor aus Thrombocyten.

6. KARACA, M., NILSSON, I. M., and HEDNER, U. Quantitative determination of platelet fibrinogen. J. Lab. Clin. Med. 77, 485, 1971. 7. KEENAN, J. P. Platelet fibrinogen. I. Quantitation using sensitized red cells. Med. Lab. Technol. 29, 71, 1972. 8. DOOLITTLE, R. F., TAKAGI, fibrinogens are identical

fibrinogen

T., and COTTRELL, B. A. Platelet and plasma gene products. Science 185, 368, 1974.

9. SOLUM, N. 0. and LOPACIUK, S. Bovine platelet proteins. III. Some properties of platelet fibrinogen. Thromb. Diath. Haemorrh. 21, 428, 1969. 10. JAMES, H. L., BRADFORD, H. R., and GANGULY, P. Platelet fibrinogen. Identity and initial observations on the mode of its degradation by plasmin. Biochim. Biophys. Acta 386, 209, 1975.

1: fj!!

SLq-PLATELET

F-I

Zc F-XIII

Vo1.8,?30.4

11. NACHMILC', R. L., MARCUS, A. J., and ZUCKER-FMKLIN, D. Immunologic studies of proteins associated with subcellular fractions of normal human platelets. J. Lab. Clin. Med. 69, 651, 1967. 12. NACHMAN, R. L. and IMARCUS,A. J. Immunological studies of proteins associated with the subcellular fractions of thrombasthenic and afibrinogenaemic platelets. Brit. J. Haematol. 15, 181, 1968. 13. DAY, H. J. and SOLUM, N. 0. Fibrinogen associated with subcellular platelet particles. Stand. J. Haematol. 10, 136, 1973. 14. BOHN, H., HAUPT, H., and KRANZ, T. Die molekulare Struktur der fibrinstabilisierenden Faktoren des Manschen. Blut 25, 235, 1972. 15. SCHWARTZ, M. L., PIZZO, S. V., HILL, R. L., and MCKEE, P. A. Human factor XIII from plasma and platelets. J. Biol. Chem. 248, 1395, 1973. 16. BROEKMAN, M. J., WESTMORELAND, N. P., and COHEN, P. An improved method for isolating alpha granules and mitochondria from human platelets. J. Cell Biol. 60, 507, 1974. 17. MILLER, G. L. Protein determinations for large number of samples. Analyt. Chem. 31, 964, 1959. 18. HAWIGER, J., NIEWIAROWSKI, S., GUREWICH, B., and THOMAS, D. P. Measurement of fibrinogen and fibrin degradation products in serum by staphylococcal clumping test. J; Lab. Clin. Med. 75, 93, 1970. 19. LORAND, L., URAYAMA, T., DE KIEWIET, J. W. C., and NOSSEL, H. L. Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation. J. Clin. Invest. 48, 1054, 1969. 20. KORNBERG, A. Lactic dehydrogenase of muscle. In: Methods in Enzymology, vol. 1. S. P. Colowick and N. E. Kaplan (Eds.) New York, Academic Press, 1955, p. 441. 21. BOSMANN, H. B. Identification, purification and characteristics of glycosidases of human blood platelets. Biochim. Biophys. Acta 258, 265, 1972. 22. TANGEN, O., BERMAN, H. J., and MARFEY, P. Gel filtration: A new technique for separation of blood platelets from plasma. Thromb. Diath. Haemorrh. 25, 268, 1971. 23. HOVIG, T. Release of a platelet aggregating substance (adenosine diphosphate) from rabbit blood platelets induced by saline 'extracts' of tendons. Thromb. Diath. Haemorrh. 9, 264, 1963. 24. RATNOFF, 0. D. and MENZIE, C. A new method for the determination of fibrinogen in small samples of plasma. J. Lab. Clin: Med. 37, 316, 1951. 25. LORAND, L., URAYAMA, T., ATENCIO, A. C., and HSIA, D. Y. Y. Inheritance of deficiency of fibrin-stabilizing factor (factor XIII). Amer. J. Human Genet. 22, 89, 1970. 26. MCDONAGH, J., MCDONAGH, R. P., MYLLYLA, G., and IKKALA, E. Factor XIII deficiency: a genetic study of two affected kindreds in Finland. Blood

SI_If3-PLATELET

F-I

$65

23 F-XIII

43, 327, 1971. 27. JAMES, H. L. and GANGULY, P. Identity of human platelet Biochem. Biophys. Res. Couunun. 63, 659, 1975. 28. BROEKM.%N, J. J., HANDIN, R. I.., and COHEN, P. and platelet factors 4 and XIII in subcellular platelets. Brit. J. Haematol. 31, 51, 1975. 29. KIESSELBACH, T. H. and WAGNER, R. H. human megakaryocytes by a fluorescent Acad. Sci. 202, 318, 1972.

fibrinogen.

Distribution of fibrinogen fractions of human

Demonstration of factor XIII in Ann. N. Y. antibody technique.

Factor 30. MCDOSAGH, J., MCDONAGH, R. P., DELAGE, J. M., and WAGNER, R. H. XIII in human plasma and platelets. J. Clin. Invest. 48, 940, 1969. 31. GRETTE, K. Studies on the mechanism of thrombin-catalyzed hemostatic reactions in blood platelets. Acta Physiol. Stand. 56, Suppl. 195, 10, . 1962. 32. DAVEY, M. G. and LUSCHER, E. F. Release'reactions of human platelets induced by thrombin and other agents. Biochim. Biophys. Acta 165, 490, 1968. 33. KEENAN, J. P. and SOLUM, N. 0. Quantitative studies platelet fibrinogen by thrombin. Brit. J. Haematol.

on the release of 23, 461, 1972.

34. JOIST, J. H. and NIEWIAROWSKI, S. Retention of platelet fibrin stabilizing factor during the platelet release reaction and clot retraction. Thromb. Diath. Haemorrh. 29, 679,, 1973. 35. SOLUM, N. 0. ADP-induced aggregation of washed platelets. Effects platelet and plasma fibrinogen. Stand. J. Haematol. 7, 236, 1970. 36. WALSH, P. M. 597, 1974.

Platelet

coagulant

activities

and hemostasis.

of

Blood 43,

Subcellular distribution of fibrinogen and factor XIII in human blood platelets.

THRO>IB0313 Printed RESEJ.RCH in the United SUBCELLULAR S. Lopaciuk", Vol. States 8, pp. Ajj-465, Pergamon Press, DISTRIBUTION OF FIBRINOGEN AND...
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