Immunochemical Studies of Bovine Platelet Factor V

1979 ◽  
Author(s):  
T.R. Ittyerah ◽  
R. Rawala ◽  
R.W. Colman
Keyword(s):  
Gel Filtration ◽  
Fold Increase ◽  
Single Line ◽  
Specific Antiserum ◽  
Factor V ◽  
Platelet Factor ◽  
Unit Structure ◽  
Bovine Plasma ◽  
Plasma Factor ◽  
New Knowledge

Utilizing new knowledge of the sub unit structure of bovine plasma factor V, we studied factor V derived from bovine platelets. Platelets were freed of plasma factor V by gel filtration and solubilized in 0.2% Trit on X-100. Incubation of this extract with a specific antiserum against purified plasma factor V rapidly inactivated platelet factor V. Antiserum absorbed with platelet extract failed to inactivate plasma factor V, further indicating that platelet factor V is antigeni cally related to plasmafactor V. Platelet factor V in the extract increased 4.7 fold when activated with thrombin while factor V released from gel filtered platelets by collagen was activated 7.5 fold. In contrast, an 18 fold increase inactivity was observed when diluted plasma or purified plasma V was incubated with thrombin suggesting that plrttelet factor V is partially activated during release. On immunoelectrophoresis, factor V released by collagen or extracted with Triton appeared as a single line close to the origin in contrast to plasma factor V which migrated towardth, anode. Incubation of the collagen releasate with antiserum to plasma factor V resulted in an immunoprec ipitate which when electrophoresed in SDS gave a single band with Mr=270,000, corresponding closely to the h chain of bovine plasma V, Mr =290,000. The alteration in platelet fact or V during release may facilitate its role in prothrombin conversion.

scholarly journals Factor V activity of platelets: evidence for an activated factor V molecule and for a platelet activator

Blood ◽  
1977 ◽  
Vol 49 (5) ◽  
pp. 819-834 ◽  
Author(s):  
B Osterud ◽  
SI Rapaport ◽  
KK Lavine
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Factor V ◽  
Freezing And Thawing ◽  
Early Step ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Native Factor

Abstract This study was prompted by the observation that fresh platelet suspensions--prepared by gel filtration or albumin density gradient centrifugation--possessed only minimal factor V activity, whereas frozen-and-thawed platelet suspensions possessed striking factor V activity. Results of experiments with fresh suspensions suggested that unaltered platelets did not bind plasma factor V. The factor V activity of frozen-and-thawed platelet suspensions was markedly diminished after exposure to a factor V antibody, was not activated by thrombin, and was not associated with an increase in factor V antigen over that found in fresh platelet suspensions. Consequently, disruption by freezing and thawing must have resulted in the appearance of small amounts of an activated factor V molecule in platelet suspensions. Disrupted platelets were shown to activate native factor V, but an interaction between a platelet activator and traces of native factor V in fresh suspensions could not be demonstrated to account for the full activity of frozen-and-thawed suspensions. Apparently, therefore, platelets also contained an activated factor V molecule. Adding collagen, but not adenosine 5′-diphosphate to fresh platelet suspensions increased their factor V activity. Release of an activated platelet factor V molecule after exposure to collagen could represent a physiologically significant early step in hemostasis.

scholarly journals Factor V activity of platelets: evidence for an activated factor V molecule and for a platelet activator

Blood ◽  
1977 ◽  
Vol 49 (5) ◽  
pp. 819-834 ◽  
Author(s):  
B Osterud ◽  
SI Rapaport ◽  
KK Lavine
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Factor V ◽  
Freezing And Thawing ◽  
Early Step ◽  
Platelet Factor ◽  
Plasma Factor ◽  
Native Factor

This study was prompted by the observation that fresh platelet suspensions--prepared by gel filtration or albumin density gradient centrifugation--possessed only minimal factor V activity, whereas frozen-and-thawed platelet suspensions possessed striking factor V activity. Results of experiments with fresh suspensions suggested that unaltered platelets did not bind plasma factor V. The factor V activity of frozen-and-thawed platelet suspensions was markedly diminished after exposure to a factor V antibody, was not activated by thrombin, and was not associated with an increase in factor V antigen over that found in fresh platelet suspensions. Consequently, disruption by freezing and thawing must have resulted in the appearance of small amounts of an activated factor V molecule in platelet suspensions. Disrupted platelets were shown to activate native factor V, but an interaction between a platelet activator and traces of native factor V in fresh suspensions could not be demonstrated to account for the full activity of frozen-and-thawed suspensions. Apparently, therefore, platelets also contained an activated factor V molecule. Adding collagen, but not adenosine 5′-diphosphate to fresh platelet suspensions increased their factor V activity. Release of an activated platelet factor V molecule after exposure to collagen could represent a physiologically significant early step in hemostasis.

scholarly journals Subcellular Localization and Secretion of Factor V by Human Platelets

1977 ◽  
Author(s):  
D.D. Pifer ◽  
R.W. Colman ◽  
C.Mcl. Chesney
Keyword(s):  
Molecular Weight ◽  
Subcellular Localization ◽  
Gel Filtration ◽  
Molecular Size ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Factor V ◽  
Platelet Factor ◽  
Dense Granules ◽  
Plasma Factor

Previous studies have suggested that factor V in platelets is derived from plasma. In order to test this hypothesis, we studied the subcellular localization, release by aggregating agents, molecular size, and stability of factor V in human platelets. When platelet homoge-nates were fractionated on sucrose density gradient the factor V activity distributed primarily into the granules (0.076 units factor V activity/mg protein) with less amounts in the membrane fraction (0.014 units/mg protein) and virtually none in the dense granules or cytosol. Collagen released 79-3% of total platelet factor V clotting activity while ADP and epinephrine failed to liberate factor V activity, further supporting significant localization in the lysozomal granules rather than the dense granules. Platelet factor V can be solubilized by 0.1% Triton and has an apparent molecular weight of 470,000 as determined by 4% agarose gel filtration. In contrast, plasma factor V in the presence or absence of Triton has a molecular weight of 300,000. Factor V activity from the platelet homogenate exhibited biphasic decay at 37°C, suggesting more than one form, whereas plasma factor V shows a first order decay curve. These data suggest that platelet factor V is predominantly localized in the granules and is an intrinsic constituent of the platelet with different properties from plasma V.

Human platelets contain forms of factor V in disulfide-linkage with multimerin

2004 ◽  
Vol 92 (12) ◽  
pp. 1349-1357 ◽  
Author(s):  
Nola Fuller ◽  
Shilun Zheng ◽  
Frédéric Adam ◽  
Samira Jeimy ◽  
Ian Horsewood ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Human Platelets ◽  
Factor V ◽  
Single Chain ◽  
Platelet Factor ◽  
Disulfide Linkage ◽  
Factor Va ◽  
Plasma Factor ◽  
Large Platelet ◽  
Large Factor

SummaryFactor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factorV is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage.The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

scholarly journals Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2180-2190
Author(s):  
MD Rand ◽  
M Kalafatis ◽  
KG Mann
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Factor V ◽  
Platelet Factor ◽  
Factor Va ◽  
Plasma Factor

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

Human Platelets Contain Forms of Factor V in Disulfide-Linkage with Multimerin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1933-1933
Author(s):  
Catherine P.M. Hayward ◽  
Nola Fuller ◽  
Shilun Zheng ◽  
Frederic Adam ◽  
Samira Jeimy ◽  
...  
Keyword(s):  
Factor V ◽  
Single Chain ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Disulfide Linkage ◽  
Thrombin Cleavage ◽  
Factor Va ◽  
Plasma Factor ◽  
Large Factor ◽  
V Antigen

Abstract Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric α-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains, were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Because unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that all unusually large factor V in platelets was associated with multimerin and it remained associated in 0.5 M salt. Multimerin immunodepletion of the normal pooled platelet lysate removed 100 ± 0% of multimerin and 47.0 ± 2.4% of total factor V antigen, whereas sham immunodepletion removed 12.0 ± 3.0 % of multimerin and 4.0 ± 4.0% of factor V antigen (means ± 1 S.D. for 3 experiments). Analyses of serial factor V immunopurified samples indicated that platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The suggestion that only a subpopulation of multimerin was covalently linked to factor V was consistent with the estimated 17 fold molar excess of multimerin subunits to factor V molecules in platelets. The disulfide-linked complexes of multimerin and factor V in platelets, that are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Multimerin could function to hold about half of the platelet pool of factor V in covalent and noncovalent linkages, until granule release occurs and thrombin cleavages liberate factor Va for prothrombinase assembly on the platelet surface, akin to the way supporting scaffolds hold pieces of plastic models in a unit until their removal for model assembly is desired.

scholarly journals Tridegin, a new peptidic inhibitor of factor XIIIa, from the blood-sucking leech Haementeria ghilianii

1997 ◽  
Vol 324 (3) ◽  
pp. 797-805 ◽  
Author(s):  
Sarah FINNEY ◽  
Lisa SEALE ◽  
Roy T. SAWYER ◽  
Robert B. WALLIS
Keyword(s):  
Tissue Transglutaminase ◽  
Gel Filtration ◽  
Factor Xa ◽  
Enzyme Concentration ◽  
Factor Xiiia ◽  
Salivary Gland Extract ◽  
Platelet Factor ◽  
Exchange Cation ◽  
Blood Sucking ◽  
Plasma Factor

1. Crude salivary gland extract of the giant Amazon leech, Haementeria ghilianii, contains an inhibitor of plasma factor XIIIa. 2. The inhibitory agent was purified to homogeneity by anion-exchange, cation-exchange, gel-filtration and reverse-phase chromatography to yield a single band on SDS/PAGE with an apparent molecular mass of 7.3 kDa. It has been named tridegin. 3. Micro-sequencing of proteolytic fragments showed tridegin to be a peptide of 66 amino acids. The sequence is unique with little similarity to other leech-derived proteins. 4. Inhibition of plasma factor XIIIa activity was confirmed by four independent methods: tridegin increased the solubility of fibrin clots in urea, inhibited ammonia produced from the incorporation of ethylamine into casein, inhibited the incorporation of 5′-(biotinamido)pentylamine into casein and prevented γ-dimer formation in clotting fibrinogen. 5. The IC50 of tridegin (approx. 9.2 nM) is very close to the concentration of factor XIIIa used in the assay and in fact depends on its concentration. This is the most potent inhibitor of factor XIIIa yet described. 6. Tridegin also inhibits platelet factor XIIIa (factor XIIIAa) with a similar potency to that of the plasma enzyme. 7. Tridegin also inhibits tissue transglutaminase but with lower potency and independently of the enzyme concentration. 8. Tridegin appears to be specific for transglutaminases, since it has no effect on the coagulation times of human plasma, on thrombin or factor Xa. Moreover it has no effect on other thiol-containing enzymes and has no ability to digest fibrinogen or cleave the isopeptide substrate, l-γ-glutamyl-4-nitroanilide.

Endocytosis and storage of plasma factor V by human megakaryocytes

2005 ◽  
Vol 94 (09) ◽  
pp. 585-592 ◽  
Author(s):  
Youko Suehiro ◽  
Dragoslava Kika Veljkovic ◽  
Nola Fuller ◽  
Yasuaki Motomura ◽  
Jean Marc Massé ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Direct Evidence ◽  
Human Platelet ◽  
Factor V ◽  
Platelet Factor ◽  
Exogenous Protein ◽  
Plasma Factor ◽  
And Storage

SummaryFactor V is an essential coagulation cofactor that circulates in plasma and platelet α-granules where it is stored complexed to multimerin 1 (MMRN1). To gain insights into the origin and processing of human platelet factor V, and factor V-MMRN1 complexes, we studied factor V in cultured megakaryocytes. Factor V mRNA was detected in all megakaryocyte cultures. However, like albumin, IgG and fibrinogen, factorV protein was detectable only in megakaryocytes cultured with exogenous protein. The amount of factor V associated with megakaryocytes was influenced by the exogenous factorV concentration. Similar to platelet factor V, megakaryocyte factor V was proteolyzed and complexed with megakaryocyte-synthesized MMRN1. With secretagogues, megakaryocytes released factorV, IgG, fibrinogen and MMRN1. Immunofluorescent and electron microscopy confirmed factorV uptake by endocytosis and its trafficking to megakaryocyte α-granules. These data provide direct evidence that human megakaryocytes process plasma-derived factor V into α-granules and generate factor V-MMRN1 complexes from endogenously and exogenously synthesized proteins.

Human Plasma And Platelet Factor V Levels As Measured By Radioimmunoassay

1981 ◽  
Author(s):  
P B Tracy ◽  
J M Peterson ◽  
M E Nesheim ◽  
J A Katzmann ◽  
K G Mann
Keyword(s):  
Human Factor ◽  
Specific Activity ◽  
Human Platelets ◽  
Prolonged Storage ◽  
Factor V ◽  
Platelet Factor ◽  
Average Value ◽  
Standard Curve ◽  
Plasma Factor ◽  
Bioassay Data

Highly purified human Factor V was used for the development of a competitive double antibody radioimmunoassay (RIA) using 125I-human Factor V, burro anti-human Factor V antisera as the primary antibody and goat anti-burro antisera as the precipitating antibody. The standard curve allows the detection of as little as 20 ng of Factor V per ml of plasma. With this specific RIA for human Factor V, we have measured the level of Factor V in the plasma and platelets of normal individuals. The normal level of Factor V in plasma ranges from 4 to 14 μg per ml, with the average value equal to 7.0 ± 2.0 μg/ml (n = 64; 33 females; 31 males; 22 to 61 years of age). There appeared to be no correlation between antigen levels and age or sex. Factor V clotting assays were consistent with the RIA data for any given plasma preparation providing freshly drawn plasma was used in the bioassay. The bioassay data were quantitated based upon the specific activity of purified plasma Factor V; 1.7 units of Factor V equals 1 μg of protein. Plasma Factor V antigen levels were not affected by lyophilization of the plasma, prolonged storage of the plasma at -20°C or intentional conversion of the plasma to serum. The levels of Factor V present in washed human platelets were also determined using the RIA. Assay of washed platelets lysed in 0.2% Triton X-100 indicated that 0.6 to 0.85 μg of Factor V was present per 2.5 × 108 platelets (4400 to 6200 molecules of Factor V per platelet). This result is in marked contrast to our observations for the bovine system, where we found that bovine platelets possess approximately 400 to 800 molecules of Factor V per platelet, and plasma Factor V levels range from 30 to 50 μg per ml. In the bovine system, the platelets possess approximately 1% of the total Factor V present, while in human blood, the platelets possess as much as 10 to 15% of the total Factor V present.

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