Platelet Cofactor Research Articles

Platelets and platelet-derived factor Va confer hemostatic competence in complete factor V deficiency

AbstractWhole genome sequencing of an individual completely devoid of plasma- and platelet-derived factor V (FV) identified 167 variants in his F5 gene including previously identified and damaging missense mutations at rs6027 and Leu90Ser. Because the administration of fresh frozen plasma (FFP) prevents gastrointestinal bleeding in this individual, its effects on his plasma- and platelet-derived FV concentrations were assessed. The patient's plasma FV levels peaked by 2 hours following FFP administration and were undetectable 96 hours later. In contrast, increased platelet-derived FV/Va concentrations were observed within 6 hours, peaked at 24 hours, decreased slowly over 7 days, and originated from megakaryocyte endocytosis and intracellular processing of plasma FV. Ten days after transfusion, no thrombin was generated in a tissue factor–initiated whole blood clotting assay unless exogenous FV was added, consistent with the complete absence of plasma FV. In marked contrast, release of the patient's platelet-derived FV/Va (7% of normal) following platelet activation resulted in robust thrombin generation, similar to that in an individual with normal plasma- and platelet-derived FV concentrations. Thus, total FV deficiency can be corrected by plasma administration, which partially repletes and sustains the platelet cofactor pool, thereby highlighting the critical role of platelet-derived FV/Va in ensuring hemostatic competence.

Anticoagulant Modulation of Blood Cells and Platelet Reactivity by Garlic Oil in Experimental Diabetes Mellitus

Multiple blood cell types and metabolic pathways involved in the modulation of platelet reactivity were investigated in streptozotocin-induced diabetic rats treated with garlic oil. Platelet counts of diabetic rats treated with garlic oil were significantly (P<0.01) reduced as compared to diabetic control rats. Garlic oil also increased the leucocyte counts of diabetic rats as compared to diabetic control rats. The significant (P<0.001) decreases by garlic oil of plasma concentration factors, V, VII, VIII: C, IX and X in diabetic rats may be interpreted to mean that there was a modulation of factor VII similar to that brought about by thrombin on factors V and VIII: C. This reversal of hypercoagulation through integrated biochemical reaction is suggestive of multicellular modulation of platelet reactivity, erythrocytes and neutrophils and the functional interactions between plasma coagulation factors and platelet cofactors.

The Mechanism of Inactivation of Human Platelet Factor Va from Normal and Activated Protein C-resistant Individuals

The inactivation of human platelet factor Va by activated protein C (APC) was analyzed by functional assessment of cofactor activity and Western blotting analysis to visualize the factor Va fragments accompanying proteolysis. Platelets were treated with thrombin to facilitate both their activation as well as the release and further activation of platelet factor Va, followed by APC addition. The rates of inactivation were donor-dependent such that 15-60% of the initial cofactor activity was lost within 5 min of APC addition with as much as 10-20% of the activity still remaining after 2 h of incubation. Western blot analysis using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the factor V molecule suggested that the factor Va activity resistant to APC inactivation was due to residual heavy chain. Furthermore, in contrast to studies with normal plasma-derived factor Va, two possible cleavage mechanisms could explain the platelet factor Va fragments observed. APC can cleave platelet factor Va initially at Arg506, with subsequent cleavages occurring at Arg306 and Arg679. Alternatively, APC can cleave at Arg306 initially, with further cleavage at Arg679 then at Arg506 or at Arg506 followed by cleavage at Arg679. Similar results were obtained if platelets were removed from the inactivation mixtures and phospholipid vesicles were used to supply the membrane surface required for inactivation, suggesting that the order of platelet factor Va peptide bond cleavage or the amount of cofactor activity remaining was not altered by either of these surfaces. Thus, APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. Analogous protocols were used to study an individual heterozygous for the Arg506-->Gln506 mutation (Factor V Leiden, Factor VR506Q). With respect to the mutant platelet factor Va in the presence of APC, > 70% of the initial cofactor activity remained after 1 min, with 30% activity still remaining after 2 h. As seen in studies of the APC-catalyzed inactivation of plasma factor VaR506Q, proteolysis of the mutant platelet factor Va confirms that even though cleavage at Arg306 will occur in the absence of cleavage at Arg506, the rate of inactivation is slower. Collectively these data suggest that when compared to normal plasma factor Va, differences in normal platelet factor Va which define: 1) whether the heavy chain is susceptible to cleavage at Arg306 or Arg506 and 2) the extent to which it is cleaved initially at Arg306, in contrast to cleavage of Arg506, will define both the extent and rate of inactivation.

X. Platelets, Phospholipids and Platelet Cofactors

X. Platelets, Phospholipids and Platelet Cofactors

Asialofibrinogen Supports Platelet Aggregation and Adhesion to Glass

Fibrinogen is required for the initial phase of platelet aggregation induced by ADP and epinephrine and the adhesion of platelets to glass. Since the carbohydrate moiety of glycoproteins has been implicated in their interactions with cell surfaces, I investigated the role of fibrinogen's sialic acid in these platelet cofactor functions. Plasminogen-free human fibrinogen (which had no detectable factor VIII/von Willebrand factor antigen by electroimmunoassay) was treated with 0.1 U/ml CI. perfringens neuraminidase at pH 6.2 for 3 hr at 37°C and then further purified by gel filtration and glycine precipitation. The neuraminidase had no detectable protease activity against casein, with a lower limit of sensitivity of 20 mU trypsin/U neuraminidase. Desialylation was greater than 95 % complete, since the treated fibrinogen had

Laboratory analysis of clotting - factor components concentrated for therapeutic use

A laboratory study was made of Factor IX preparations used or produced for therapeutic use. They contained large amounts of prothrombin and autoprothrombin III (F-X). In solution, ready for use, prothrombin and autoprothrombin III remained at the original concentration for about 3 weeks at 4° C without developing thrombin or autoprothrombin C (F-X a) activity. One of the products was dissolved in 2.5 M glycine solution at 4° C. Autoprothrombin C activity developed and all the prothrombin converted to thrombin over a period of 70 days. The thrombin activity generated after the prothrombin was modified. The Factor IX activity in these preparations was easily measured by the platelet cofactor assay. It was not as stable as the prothrombin or autoprothrombin III. The same conclusion about stability was reached from prothrombin utilization experiments with plasma from a hemophilia B subject. The Factor IX preparations which were studied are suitable reagents for the assay of platelet factor 3. Purified bovine prothrombin complex prepared in this laboratory contained very little platelet cofactor (Factor VIII) activity nor did it contain platelet cofactor II (Factor IX) activity. For this reason, the purified bovine prothrombin complex is a satisfactory reagent for the assay of Factor VIII concentrates. Platelet cofactors have a synergistic procoagulant effect with platelet factor 3 in the formation of autoprothrombin C from its precursor.

Blood Coagulation

SummaryPreparations of platelet factor 3 accelerated the conversion of purified autoprothrombin III (F-X) to autoprothrombin C (F-X?). The reaction was slow and the yields were low. Purified Hageman factor or purified autoprothrombin II (F-IX?) functioned as platelet cofactors. Each increased the yield of autoprothrombin C from purified autoprothrombin III above that obtained with platelet factor 3 alone. Hageman protein thus has a role in the direct acceleration of the first basic reaction in blood coagulation. Purified prothrombin was made refractory to the two-stage analytical reagents with Hageman factor.

Blood-Clotting Enzymology

Abstract During the past quarter century perhaps no single phenomenon has attracted so much attention in biology and medicine as blood clotting. It plays a role in numerous clinical conditions where it was not taken into account previously. Knowledge of the basic mechanisms recently passed through a tumulous stage of growth, but is now delineated in terms of simple enzymology. Most of the components have been obtained in purified form. There are three main events: the formation of autoprothrombin C (F-Xa, thrombokinase), the formation of thrombin, and the formation of fibrin. Prothrombin itself is a more marvelous protein than was predicted. It is a protein aggregate that contains the precursor of thrombin, the precursor of autoprothrombin C, as well as accessory protein. When blood coagulation is initiated by tissue injury, large amounts of autoprothrombin C activity arise in the presence of calcium ions and tissue thromboplastin. When there is no tissue injury, small amounts of autoprothrombin C are produced by platelet cofactor I (F-VIII, AHF) which functions with the lipids of platelets. Autoprothrombin C is the enzyme which produces thrombin activity, and being a part of the prothrombin aggregate it is in close proximity to the specific bond(s) that must be split to get thrombin activity. Although autoprothrombin C alone is sufficient to produce thrombin, the reaction is greatly accelerated by plasma Ac-globulin (F-V), lipids from platelets, and calcium ions. The acceleration produced by components functioning in pairs and relays makes possible the essential integration with the anatomic location of resources. Tissue materials pair with plasma resources to produce autoprothrombin C, and then they pair with another plasma component (Ac-globulin) to make autoprothrombin C function. In the absence of tissue injury, platelet materials pair with plasma resources to produce autoprothrombin C, and then platelets again work with another plasma component (Ac-globulin) to support the function of autoprothrombin C. Platelets are essential because their lipids are needed for the formation of autoprothrombin C, as well as for accelerating its function. Whatever involves platelets can be the stimulus to accelerate blood clotting; this includes Hageman factor. Platelets are the true point of beginning for the first step in blood clotting and are involved in the second and third. Vitamin K deficiency, or counteraction of Vitamin K by Dicumarol reduces prothrombin synthesis by the liver. As a consequence the precursor of thrombin, the precursor of autoprothrombin C, and the accessory protein of the molecular aggregate which constitutes prothrombin is reduced in concentration. It has been found possible to produce abnormal conditinos experimentally and have relatively more of the precursor of thrombin released to the blood than is normally associated with the precursor of autoprothrombin C. Up to the point of incorporation to form a single prothrombin molecule, the synthesis of subunits proceeds by separately operating mechanisms.

Physiology of blood coagulation: advances, problems and present status.

Advances have been made in the purification of the molecules known to be concerned in blood clotting, and on the foundations of this work it is possible to state that the basic enzymology consists of three major events. In sequence there occurs: 1. the formation of autoprothrombin C (F-Xa, thrombokinase), 2. the formation of thrombin by autoprothrombin C, and 3. the formation of fibrin by thrombin through limited proteolysis. In this laboratory prothrombin was first purified and was found to be an aggregate which contains the precursor of thrombin (prethrombin) and the precursor of autoprothrombin C (autoprothrombin III). It was discovered that purified prothrombin could be activated by simply placing it in 25% sodium citrate solution. In this activation it first dissociates to form mainly prethrombin and autothrombin III. Next the autoprothrombin III converts to autoprothrombin C, and this enzyme then converts the prethrombin to thrombin. Sound evidence for the first two major steps in blood clotting was thus obtained by carefully avoiding all clotting factors real or imagined. Only prothrombin and 25% sodium citrate was needed as starting material. This achievement introduced clarification, simplification, and the degree of certainty which was very valuable as a foundation on which to place the superstructure concerned with accessory factors. The reactions in 25% sodium citrate solutions are slow, but the accessory factors used in our normal physiology accelerate the prototype reactions found to be essential. These accessory substances are located in fixed tissue cells, in platelets, and in plasma. The formation of autoprothrombin C, the first main event in blood clotting, is brought about rapidly by calcium ions and thromboplastin or not quite so rapidly by calcium ions, platelet lipids, and platelet cofactor I (F-VIII, AHF). The function of autoprothrombin C is to bring about the second main reaction; namely, the generation of thrombin activity. To obtain thrombin rapidly with autoprothrombin C it is necessary to have the Ac-globulin (F-V) of plasma and the lipids of platelets. Incidentally the lipids of thromboplastin can substitute for those of platelets in the conversion of prethrombin to thrombin. The lipids are surface active agents and the Ac-globulin protein determines the substrate specificity of the autoprothrombin C. In the absence of tissue extracts platelets are practically essential in the first two of the three main reactions in blood clotting. The recurring theme is the simultaneous participation of lipid and protein functioning as two molecular entities from two anatomic compartments. Injury is the natural way for substances in different compartments to be released and brought together.

Isolation and some properties of prethrombin and autoprothrombin III

Purified bovine prothrombin preparations were dissociated by digestion with thrombin. The reaction mixture was separated to obtain an inhibitor, the precursor of autoprothrombin C called autoprothrombin III, and the precursor of thrombin called prethrombin. Prethrombin was purified and found to have an isoelectric point at pH 5.5 and in the ultracentrifuge s 0 20, w = 3.9. It contained 75 more amino acid residues than purified thrombin. Autoprothrombin III was also purified. The observed isoelectric point was pH 4.7, and s 0 20, w = 3.4. After the separation and purification of prethrombin and autoprothrombin III, it was possible to study the activation requirements of each one independently of the other. Autoprothrombin III readily converted to autoprothrombin C in the presence of tissue thromboplastin and calcium ions, but other suitable conditions for activation were also found. The formation of autoprothrombin C was observed to occur slowly in the presence of calcium ions and platelet cofactor I. It is therefore concluded that rapid clotting of blood with tissue extracts as compared with platelets plus plasma is due to the vastly greater quantity of autoprothrombin C produced with tissue extracts than with platelets plus plasma. The conversion of prethrombin to thrombin required only autoprothrombin C; however, the reaction was accelerated by the simultaneous addition of purified Ac-globulin, lipids, and calcium ions. A sequence in blood coagulation occurs in three major steps; namely, (a) the formation of autoprothrombin C, (b) the formation of thrombin, and (c) the formation of fibrin. Purified autoprothrombin III corrected the prolonged one-stage prothrombin time of Stuart plasma and also of “factor VII” deficient plasma.

Concentration of Platelet Cofactor I (Factor VIII) from Bovine Serum*

SummaryA method for obtaining concentrates of platelet cofactor I (factor VIII) from ether extracted bovine serum has been developed. Platelet cofactor I is inactivated during the coagulation process by undergoing a complex formation, which is broken by ether extraction.In each of 13 preparations from 1,000 ml of extracted serum an average total activity of 3,055 platelet cofactor I units was isolated. The specific activity was 4,760 units per milligram tyrosine and 138 units per milligram protein (Biuret.)In the paper electrophoresis the fractions were homogeneous. Using immunoelectrophoretic techniques three bands were found against anti-bovine serum from rabbits. The preparations activated purified prothrombin in the presence of platelets, Ac-globulin and calcium ions. They substituted in the thromboplastin generation test for adsorbed plasma and corrected the delayed thromboplastin generation of hemophilia A plasma. They also corrected the prolonged recalcification times of hemophilia A plasma. The preparations lost their activity upon storage at room temperature and in the icebox rapidly. Also freeze drying resulted in a complete loss of activity.

THE PURIFICATION AND PROPERTIES OF PLATELET COFACTOR I (FACTOR VIII).

THE PURIFICATION AND PROPERTIES OF PLATELET COFACTOR I (FACTOR VIII).

The Purification and Properties of Platelet Cofactor I (Factor VIII)

The Purification and Properties of Platelet Cofactor I (Factor VIII)

Platelet Cofactors as Plasma Components of the Intrinsic Blood Clotting Mechanisms

SummaryThe method for the assay for platelet cofactors has been overhauled to make allowance for an adequate and uniform supply of Ac-globulin. Thrombin is added at zero time to reduce variability of response in the activation of prothrombin. The potency of purified platelet cofactor I is about twice that of platelet cofactor II (autoprothrombin II). The total mass of platelet factor 3 plus platelet cofactor I or platelet cofactor II is much greater in the conversion of a given amount of purified prothrombin to thrombin than when brain thromboplastin is used.In plasma and serum from the human being barium carbonate adsorbs platelet cofactor II, and ether extraction frees activity that is not adsorbed on barium carbonate before ether extraction. The extract contains an inhibitor(s) of prothrombin activation. The inhibition is strong in the intrinsic as well as the extrinsic prothrombin activation mechanisms. In the case of bovine plasma barium carbonate adsorption leaves the plasma richer in platelet cofactor activity and in that respect it differs from the plasma from human beings. Dog plasma and serum is comparable to that from man than the bovine species.The lipids extracted from plasma or serum with ether are predominantly anticoagulant. Since platelet cofactor I activity disappears during the clotting of blood, we think that a function of platelet cofactor I is to be the receptor for an anticoagulant. Then as autoprothrombin II arises from prothrombin it serves as a procoagulant and is not retarded by the anticoagulant neutralized by platelet cofactor I.

Antithrombin II Anticoagulant (Autoprothrombin II-A)

SummaryAutoprothrombin II-A activity frequently develops when the autoprothrombin II fraction of prothrombin is dried from the frozen state. When the anticoagulant activity develops, the N-terminal amino acid arginine is found, but not when the anticoagulant activity does not develop. The anticoagulant autoprothrombin II-A retarded the conversion of prothrombin to thrombin when three different procoagulants were used; namely, platelet cofactor I, autoprothrombin II, and lung extract. The procoagulant power of the platelet cofactor I is most easily depressed. It is postulated that autoprothrombin II-A activity develops when prothrombin activates and that platelet cofactor I is a receptor of the anticoagulant. In that way the other procoagulants are not ordinarily inhibited by the autoprothrombin II-A. As a further extension of this speculation, attention is brought to the idea that autoprothrombin II-A could be present in certain hemorrhagic diseases. In the PTA patient there is no autoprothrombin II and most likely no autoprothrombin II-A. The close chemical relationship of these two prothrombin derivatives thus may account for the multiple deficiency.

Purified Platelet Cofactor I and Purified Autoprothrombin II in Thromboplastin Generation Test

SummaryPurified bovine platelet cofactor I substituted for adsorbed plasma in the thromboplastin generation test when Ac-globulin was also supplied. Only 70 gamma/ml were needed. Purified bovine autoprothrombin II substituted for serum in the test. Only 9 gamma were equivalent to serum. Platelet cofactor I thus takes the place of the antihemophilic factor and autoprothrombin II substitutes for PTC and Stuart-Prower factor. We regard the autoprothrombin II preparation used as a single component derived from prothrombin.

Some Properties of Platelet Cofactor I Concentrates of Bovine Origin

SummaryPlatelet cofactor I preparations from bovine plasma were studied from the viewpoint of stability in the presence of chemicals selected strategically. The preparations were of high quality having about 86 per cent of the protein as one component by ultracentrifuge analysis. The activity was easily destroyed with hydrogen peroxide, sodium sulfite, phenylhydrazine, formalin, acetone, and by the process of freeze drying. The activity was not lost in the presence of oxygen, ascorbic acid, cysteine, merthiolate, parachloromercuribenzoate or 0.5 per cent phenol. In the native plasma the activity is not adsorbed on barium carbonate, barium sulfate, or magnesium hydroxide. Nor is it destroyed by ether; but, in purified form it is adsorbed on these adsorbants and is destroyed by ether. In the purified preparations the activity was not adsorbed on IRC-50 resin thus making it possible to use this adsorbant to remove any thrombin that might be added to a platelet cofactor I preparation. By this technic it was possible to show that thrombin alone does not easily destroy platelet cofactor I activity.

Biophysical Studies on Platelet Cofactor I Preparations

SummaryPreparations of bovine platelet cofactor I have been examined according to biophysical methods. In the ultracentrifuge the patterns reveal a major component comprising 86% of the total and two faster moving minor components. The sedimentation constants are 6.65, 10.0, and 21.2 Svedberg units. The diffusion constant is 3.30 × 10−7 cm2 sec−1. Associating this value with the main constituent, the molecular weight is found to be 196,000. A molecular axial ratio of 12 is also obtained from these data. An axial ratio of 8 is indicated from viscosity data. The extinction coefficient at 2800 Å is 14.8.

Prothrombin Utilization Following Addition of Platelet Cofactor I Concentrates to Hemophilic Plasma

SummaryProthrombin is utilized in hemophilia B blood when a platelet cofactor I concentrate is added. It is not well utilized when the same preparation is added to hemophilia B plasma, hemophilia A plasma, or hemophilia A blood. Platelet factor 3 concentrates did not promote prothrombin utilization. Mixtures of platelet cofactor I and platelet factor 3 (threone) give prothrombin utilization under all conditions tested in hemophilia A and B.

Activation of prothrombin with thrombin.

Mixtures were made consisting of purified prothrombin as substrate (1200 u/ml) and purified thrombin as activator (83 u/ml). The calcium concentration was 0.027 m and the pH 7.2. During an observation time of 90 minutes prothrombin activates and some thrombin is formed. If purified platelet factor 3, or purified platelet cofactor I, or the lipid portion of platelet factor 3, or a certain lipid preparation are also added separately all of the prothrombin substrate becomes thrombin. Each substance is a procoagulant in the sense of functioning in support of thrombin as activator. Each procoagulant is more effective together with Ac-globulin, and even with the latter present sphingosine inhibits the activation of prothrombin by thrombin.