The Importance of Activation of Antihemophilic Globulin and Proaccelerin by Traces of Thrombin in the Generation of Intrinsic Prothrombinase Activity

Abstract There are two major time-consuming steps in intrinsic clotting in vitro. The importance of the first step—the triggering of intrinsic clotting through the generation of activation product (AP) activity—has been appreciated for several years. This paper has been concerned with the delineation of the second important time-consuming step—the generation of a trace of thrombin, which by activating both anti-hemophilic globulin (AHG, factor VIII ) and proaccelerin (factor V ), shifts the intrinsic clotting process into high gear. Data have been presented which indicate that when plasma contains AP, activated AHG (AHG'), activated proaccelerin (accelerin ) and free platelet factor 3-like activity, all of the remaining reactions required to generate powerful intrinsic prothrombinase activity take place within 7 to 12 seconds after recalcification. It may well be that AHG and proaccelerin must be activated by minute traces of thrombin before they can participate effectively in the generation of intrinsic prothrombinase activity.

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The Natural Course of Defibrination Syndrome Caused by Echis Colorata Venom in Man

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649181 ◽

1974 ◽

Vol 31 (03) ◽

pp. 420-428 ◽

Cited By ~ 3

Author(s):

M Fainaru ◽

S Eisenberg ◽

N Manny ◽

C Hershko

Keyword(s):

Factor Viii ◽

Blood Coagulation ◽

Major Bleeding ◽

Renal Damage ◽

Specific Treatment ◽

Natural Course ◽

Factor V ◽

Thrombocyte Count

SummaryThe natural course of defibrination syndrome caused by Echis colorata venom (ECV) in five patients is reported. All patients developed afibrinogenemia within six hours after the bite. Concomitantly a depression in factor V was recorded. Factor VIII and thrombocyte count in blood were normal in most patients. In the light of the known effects of ECV on blood coagulation in vivo and in vitro it is concluded that the afibrinogenemia is due to intravascular clotting.Four patients had transient renal damage, manifested by oliguria, azotemia, albuminuria and cylindruria, ascribed to microthrombi in the renal glomeruli.After the bite, the natural course was benign, no major bleeding was observed, and all signs of coagulopathy reverted to normal within 7 days. Therefore we recommend no specific treatment for this condition. In the case of heavily bleeding patients, administration of antiserum against ECV and/or heparin should be considered.

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Studies on the Pathogenesis of the Generalized Shwartzman Reaction

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655560 ◽

1964 ◽

Vol 12 (02) ◽

pp. 471-483 ◽

Cited By ~ 13

Author(s):

F Rodríguez-Erdmann

Keyword(s):

Clotting Factor ◽

Factor V ◽

Clotting System ◽

Platelet Factor ◽

Trigger Mechanism ◽

Haemorrhagic Diathesis ◽

Shwartzman Reaction ◽

Ca Ions

SummaryThe rôle of the clotting system in the pathogenesis of the generalized Shwartzman reaction (gSr) has been stressed in recent years. The clotting system is activated ubiquitously and as a result of it, fibrin is deposited intravascularly and a haemorrhagic diathesis develops. Evidence is presented herein, that endotoxin does not activate purified prothrombin, nor does endotoxin influence the convertion of prothrombin when it is activated in the presence of purified platelet-factor 3 (or caephalin) purified Ac-G (factor V) and Ca-ions.The trigger mechanism of the gSr also seems to be in the so-called prephase of clotting mechanism. Data are presented, which show that endotoxin activates the Hageman factor in vitro. The importance of this clotting factor and of platelet-factor 3 is discussed. Also the rôle played by the RES and cardiodynamic and vascular components are taken in consideration in the discussion.

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Feiba Immuno: A Preparation with Factor Eight Inhibitor Bypassing Activity

10.1055/s-0039-1682501 ◽

1977 ◽

Author(s):

F. Elsinger

Keyword(s):

Factor Viii ◽

Factor Xa ◽

Coagulation Factors ◽

Active Principle ◽

Factor Viii Inhibitor ◽

Factor V ◽

In Vitro Experiments ◽

Factor X ◽

Viii Inhibitor

FEIBA IMMUNO is a preparation in which a new activity is generated capable of bypassing factor VIII. The preparation which is used to treat patients with inhibitors (especially inhibitors to factor VIII) is standardized in FEIBA units, i.e. in terms of its in vitro capacity to shorten the activated PTT of a factor VIII inhibitor plasma.It could be concluded from different in vitro experiments that none of the classic’ activated coagulation factors is responsible for the factor VIII bypassing reaction; FEIB-activity seems to be correlated to a new complex of coagulation factors.To get an answer to the question which coagulation factors are essential for FEIB-activity, we tried to generate this activity from different deficient plasmas; from these experiments the following conclusions could be drawn:, the presence of at least factors VII, IX, and X is essential for the generation of the molecular species responsible for factor VIII as well as factor X bypassing activity, but factor V is not bypassed. This activity is not factor Xa itself. Factors VIII and V are not necessary for the generation of this active principle, but factor V is finally needed for its bypassing action.

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Most Factor VIII B Domain Missense Mutations Are Unlikely to Be Causative Mutations for Hemophilia A: Implications for Factor VIII Genetic Analysis

Blood ◽

10.1182/blood.v112.11.513.513 ◽

2008 ◽

Vol 112 (11) ◽

pp. 513-513

Author(s):

Kyoichi Ogata ◽

Steven W. Pipe

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Hemophilia A ◽

Coagulation Factor ◽

Causative Mutation ◽

Factor V ◽

Missense Mutations ◽

Severe Hemophilia ◽

Single Chain

Abstract Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII). FVIII is synthesized as a single-chain polypeptide of approximately 280 kDa with the domain structure A1-A2-B-A3-C1-C2. Whereas the A and C domains exhibit ~40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein and is dispensable for FVIII cofactor activity. Missense mutations in the FVIII B domain have been described in patients with variable phenotypes of hemophilia A. According to the NCBI SNPs (single nucleotide polymorphism) database, 22 SNPs are reported within FVIII, 11 of which occur within the B domain. FVIII B domain variant D1241E has been reported as a missense mutation associated with mild or severe hemophilia A, yet this mutation is also present in the NCBI SNPs database. We hypothesize that D1241E and most other reported B domain missense mutations are not the causative mutation for hemophilia A in these patients but represent SNPs or otherwise non-pathologic mutations. To investigate this, we analyzed 7 B domain missense mutations that were previously found in hemophilia A patients (T751S, V993L, H1047Y, D1241E, T1353A, P1641L and S1669L). Comparative analysis showed that the amino acids at these positions are not conserved in all species and in some cases, the amino acid substitution reported in hemophilia patients is represented in the native sequence in other species. Analysis with PolyPhen Software showed that only H1047Y mutation was considered as “possibly damaging”, while the others were considered as “benign”. To investigate this further, we constructed seven plasmid vectors containing these B domain missense mutations. The synthesis and secretion of FVIII wild-type (WT) and these seven mutants were compared after transient DNA transfection into COS-1 monkey cells in vitro. Analysis of the FVIII clotting activity and antigen levels in the conditioned medium demonstrated that all mutants had FVIII activity and antigen levels similar to FVIII WT. Further, FVIII WT, H1047Y and D1241E mutants were introduced into a FVIII exon 16 knock-out mouse model of hemophilia A by hydrodynamic tailvein injection in vivo. The mouse plasma was analyzed at 24 hrs for activity and antigen expression. Mutants H1047Y and D1241E expressed at 211 mU/mL and 224 mU/mL activity with FVIII antigen levels of 97 ng/mL and 118 ng/mL, respectively, similar to FVIII WT. These results suggested that H1047Y and D1241E mutants did not lead to impairments in secretion or functional activity. We conclude that most missense mutations within the FVIII B domain would be unlikely to lead to severe hemophilia A and that the majority of such missense mutations represent polymorphisms or non-pathologic mutations. Investigators should search for additional potentially causative mutations elsewhere within the FVIII gene when B domain missense mutations are identified.

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A Study of the Cation-and pH-Dependent Stability of Factors V and VIII in Plasma*

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654852 ◽

1965 ◽

Vol 14 (01/02) ◽

pp. 032-051 ◽

Cited By ~ 18

Author(s):

HJ Weiss

Keyword(s):

Factor Viii ◽

Temperature Coefficient ◽

Divalent Cations ◽

Factor V ◽

Rapid Loss ◽

Edta Plasma ◽

Ph Dependent ◽

Nickel Manganese ◽

Physical Reaction

SummaryThe in vitro lability of factors V and VIII in plasma has been studied. In agreement with previous reports, an increase in anticoagulant concentration renders both factors more labile (cation-deficient decay), as does an increase in the pH above 7.3 (alkaline-decay). Calcium appears to be the plasma cation which protects factors V and VIII against in vitro loss of activity. The protection obtained by the addition of other divalent cations depended on the type of plasma used. When resin-EDTA plasma was made cation free by dialysis at 4° C and then incubated at 37° C, the rapid loss of factors V and VIII activity could be prevented by prior addition of strontium, manganese and magnesium. In oxalate plasma, nickel, manganese, cadmium and strontium were effective.The alkaline decay of both factors V and VIII is irreversible. Partial reversibility of the cation-deficient decay was demonstrated for factor V, but not for factor VIII. The temperature coefficient for both the cation-deficient and alkaline decay is 2-3, suggesting an enzymatic rather than a physical reaction.There was no evidence to implicate thrombin, plasmin or trypsin since inhibitors of these enzymes failed to modify either type of decay.

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Characterization Of The In Vitro “Factor VIII Bypassing Activity”

10.1055/s-0038-1652639 ◽

1981 ◽

Author(s):

D L Aronson ◽

J Bagley

Keyword(s):

Factor Viii ◽

Early Stage ◽

Deae Cellulose ◽

Factor Ix ◽

Factor Xii ◽

Factor V ◽

Factor X ◽

Hemostatic Effect ◽

Prolonged Aptt

The in vitro correction of the prolonged APTT of hemophilic plasma has been ascribed to an uncharacterized entity “Factor VIII Bypassing Activity.” Such products also correct the prolonged APTT plasma deficient in Factor IX, Factor X and Factor XII, but not of Factor V deficient plasma. Correction of the APTT in Factor VIII deficient plasma by early stage coagulants such as Factor XIIa, Kallikrein and Factor IXa is minimal. These results indicate that this in vitro activity acts at the level of either the activation of Factor X or the activation of prothrombin.A coagulant has been prepared from serum by barium precipitation, heparin-agarose, DEAE cellulose and high pressure liquid chromatography (HPLC). The in vitro coagulant properties are similar to “activated” prothrombin complex (Autoplex) and the biologic and chemical properties are identical to activated Factor X.Infusion of the partially purified serum coagulant into normal dogs was well tolerated and, in contrast to Factor IX concentrates, gave no signs of DIC. Infusion into bleeding hemophilic dogs had no hemostatic effect. It is concluded that a major portion of the in vitro potency of activated prothrombin concentrates is due to activated Factor X, a material which when infused has no in vivo hemostatic effect.Acknowledgments - The authors gratefully acknowledge the studies of Dr. Henry Kingdon in hemophilic dogs.

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An In Vitro Analysis of the Combination of Hemophilia A and Factor VLEIDEN

Blood ◽

10.1182/blood.v90.8.3067 ◽

1997 ◽

Vol 90 (8) ◽

pp. 3067-3072 ◽

Cited By ~ 60

Author(s):

Cornelis van ‘t Veer ◽

Neal J. Golden ◽

Michael Kalafatis ◽

Paolo Simioni ◽

Rogier M. Bertina ◽

...

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor V ◽

Severe Hemophilia ◽

Factor Viii Activity ◽

Thrombin Formation

Abstract The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor VLEIDEN mutation, which leads to the substitution of Arg506 → Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII–deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor VLEIDEN mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa⋅tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (∼6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor VLEIDEN mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor VLEIDEN concentration. When the factor VLEIDEN concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor VLEIDEN level to understand the effect of factor VLEIDEN in hemophilia A patients. The presented data support the hypothesis that the factor VLEIDEN mutation can increase thrombin formation in severe hemophilia A.

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Genotype-phenotype correlation in combined deficiency of factor V and factor VIII

Blood ◽

10.1182/blood-2007-10-113951 ◽

2008 ◽

Vol 111 (12) ◽

pp. 5592-5600 ◽

Cited By ~ 45

Author(s):

Bin Zhang ◽

Marta Spreafico ◽

Chunlei Zheng ◽

Angela Yang ◽

Petra Platzer ◽

...

Keyword(s):

Factor Viii ◽

Current Data ◽

Factor V ◽

Primary Role ◽

Phenotype Correlation ◽

Platelet Factor ◽

Phenotype Analysis ◽

The Mean ◽

The Impact ◽

Combined Deficiency

AbstractCombined deficiency of factor V and factor VIII (F5F8D) is caused by mutations in one of 2 genes, either LMAN1 or MCFD2. Here we report the identification of mutations for 11 additional F5F8D families, including 4 novel mutations, 2 in MCFD2 and 2 in LMAN1. We show that a novel MCFD2 missense mutation identified here (D81Y) and 2 previously reported mutations (D89A and D122V) abolish MCFD2 binding to LMAN1. Measurement of platelet factor V (FV) levels in 7 F5F8D patients (4 with LMAN1 and 3 with MCFD2 mutations) demonstrated similar reductions to those observed for plasma FV. Combining the current data together with all previous published reports, we performed a genotype-phenotype analysis comparing patients with MCFD2 mutations with those with LMAN1 mutations. A previously unappreciated difference is observed between these 2 classes of patients in the distribution of plasma levels for FV and factor VIII (FVIII). Although there is considerable overlap, the mean levels of plasma FV and FVIII in patients with MCFD2 mutations are significantly lower than the corresponding levels in patients with LMAN1 mutations. No differences in distribution of factor levels are observed by sex. These data suggest that MCFD2 may play a primary role in the export of FV and FVIII from the ER, with the impact of LMAN1 mediated indirectly through its interaction with MCFD2.

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Combined factor V-VIII deficiency: a case report with studies of factor V and VIII activation by thrombin

Blood ◽

10.1182/blood.v58.5.983.bloodjournal585983 ◽

1981 ◽

Vol 58 (5) ◽

pp. 983-985 ◽

Cited By ~ 1

Author(s):

MB Hultin ◽

ME Eyster

Keyword(s):

Case Report ◽

Factor Viii ◽

In Vitro Studies ◽

Factor V ◽

Coagulant Activity

A new case of combined factor V-VIII deficiency is reported with in vitro studies of factors V and VIII activation by thrombin. The normal activation of factors V and VIII demonstrated in the patient's plasma and the equivalent levels of factor VIII coagulant activity and coagulant antigen support the hypothesis that a quantitative rather than qualitative defect in factors V and VIII is present in this disorder.

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Increased Plasminogen Activity Is An Independent Predictor of Bleeding in Hemophiliacs

Blood ◽

10.1182/blood.v112.11.4516.4516 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4516-4516

Author(s):

Andrea Gerhardt ◽

Melanie Pesch ◽

Rudiger E. Scharf ◽

Rainer B. Zotz

Keyword(s):

Factor Viii ◽

Independent Predictor ◽

Coagulation Factor ◽

Prothrombin G20210a ◽

Factor V ◽

Bleeding Events ◽

Antifibrinolytic Agents ◽

Low Levels ◽

Mucosal Bleeding

Abstract Rationale and Objectives: Apart from the differences in factor VIII:C levels, it is difficult to explain the significant variation in bleeding events observed among individual patients with similar levels of FVIII:C and standard laboratory profiles. We hypothesize that additional dispositional (genetic) and expositional (non-genetic) factors of hemostasis may augment or attenuate the likelihood of bleeding symptoms in hemophiliacs. Patients and Methods: To test this hypothesis, we studied 61 patients diagnosed with hemophilia A. The following laboratory parameters were examined in multivariate analysis using a case-only analysis: genotyping for a2C807T, HPA-1 of aIIbb3, factor V G1691A, prothrombin G20210A; coagulation factor activity/antigen of fibrinogen, factor II, V, VII, VIII, IX, X, XI, XII, XIII, vWF; in-vitro bleeding time (PFA-100 closure times), and plasminogen. Results: After adjustment for age, low levels of factor VIII (p=0.0296) and increased levels of plasminogen (p=0.0415), and collagen/ADP closure times (PFA-100) (p=0.04) could be identified as independent predictors of mucosal bleeding. 10% higher activity of plasminogen increased the bleeding risk 10.4 fold. For joint bleeding low levels of fibrinogen (p=0.003) and longer collagen/epinephrine closure times (PFA-100) could be identified as bleeding predictors. Conclusion: Apart from low levels of factor VIII, increased activity of plasminogen is an independent predictor of mucosal bleeding in hemophiliacs. In consequence, it will be of importance to examine whether the critical subgroup of patients with increased plasminogen levels and mucosal bleeding can benefit from prevention with specific antifibrinolytic agents.

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