Simultaneous Occurrence of Human Antibodies Directed against Fibrinogen, Thrombin, and Factor V Following Exposure to Bovine Thrombin: Effects on Blood Coagulation, Protein C Activation and Platelet Function

1997 ◽  
Vol 77 (02) ◽  
pp. 343-349 ◽  
Author(s):  
Vibhuti D Chouhan ◽  
Raul A De La Cadena ◽  
Chandrasekaran Nagaswami ◽  
John W Weisel ◽  
Mehdi Kajani ◽  
...  
Keyword(s):  
Protein C ◽  
Simultaneous Occurrence ◽  
Factor V ◽  
Bovine Thrombin ◽  
Plasma Factor ◽  
Coagulation Tests ◽  
Coagulation Protein ◽  
Severe Epistaxis ◽  
Clotting Protein

SummaryWe describe a patient with severe epistaxis, prolonged coagulation tests and decreased plasma factor V following exposure to bovine topical thrombin. Patient IgG, but not normal IgG, showed binding to immobilized thrombin (bovine > human) and fibrinogen, and to factor V by Western blotting; the binding to thrombin was inhibited by hirudin fragment 54-65. Electron microscopy of rotary shadowed preparations showed complexes with IgG molecules attached near the ends of trinodular fibrinogen molecules. Patient IgG inhibited procoagulant, anticoagulant and cell-stimulating functions of thrombin demonstrated by inhibition of fibrinogen clotting, protein C activation and platelet aggregation; thrombin hydrolysis of S-2238 was not inhibited. The results suggest that the antibody is targeted against anion-binding exosite and not catalytic site of thrombin. Antifibrinogen antibodies have not been reported in patients exposed to bovine thrombin. There is a pressing need to re-evaluate the role of bovine thrombin as a therapeutic agent.

Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

1993 ◽  
Vol 69 (02) ◽  
pp. 124-129 ◽  
Author(s):  
Susan Solymoss ◽  
Kim Thi Phu Nguyen
Keyword(s):  
Western Blotting ◽  
Protein C ◽  
Thrombin Generation ◽  
Blood Platelets ◽  
Activated Protein C ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

Primary Thrombophilia in Mexico III: A Prospective Study of the Sticky Platelet Syndrome

2002 ◽  
Vol 8 (3) ◽  
pp. 273-277 ◽  
Author(s):  
Guillermo J. Ruiz-Argúelles ◽  
Briceida López-Martinez ◽  
Donají Cruz-Cruz ◽  
Lucía Esparza-Silva ◽  
Ma. Belén Reyes-Aulis
Keyword(s):  
Plasminogen Activator ◽  
Protein C ◽  
Factor V Leiden ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Factor V ◽  
Factor V Leiden Mutation ◽  
G20210a Mutation ◽  
Coagulation Protein ◽  
Activator Inhibitor

During an 18-month period, 10 consecutive Mexican mestizos with a clinical marker associated with a primary hypercoagulable state were studied. The assessment of the sticky platelet syndrome (SPS) was done by the method described by Mammen. In addition, the activated protein C resistance phenotype, coagulation protein C activity and antigen, coagulation protein S, antithrombin III, plasminogen, tissue-type plasminogen activator activity, plasminogen activator inhibitor activity, plasminogen activator inhibitor type 1, IgG and IgM isotypes of anti-phospholipid antibodies, homocysteine levels, the factor V gene Leiden mutation, the 677 C->T mutation in the 5,10-methylen-tetrahydrofolate-reductase (MTHFR), and the G20210A polymorphism in the 3′-untranslated region of the prothrombin gene were studied. Six patients with the SPS were identified: only one displayed this as the single thrombophilic abnormality; in five others, additional thrombosis-prone conditions were found: heterozygosity for the MTHFR 677 gene mutation in five cases; and, in one case each, heterozygosity for the factor V Leiden mutation, heterozygosity for the factor II G20210A mutation, and antiphospholipid antibodies in another. Four of the six patients had a family history of thrombophilia. All patients were treated with aspirin and no new vasoocclusive episodes have been recorded. SPS in not an infrequent finding in Mexican mestizo thrombophilic patients and may contribute to thrombophilia.

scholarly journals ptFVa ( Pseudonaja Textilis Venom-Derived Factor Va) Retains Structural Integrity Following Proteolysis by Activated Protein C

2021 ◽  
Author(s):  
Mark Schreuder ◽  
Xiaosong Liu ◽  
Ka Lei Cheung ◽  
Pieter H. Reitsma ◽  
Gerry A.F. Nicolaes ◽  
...  
Keyword(s):  
Protein C ◽  
Structural Integrity ◽  
Noncovalent Interactions ◽  
Activated Protein C ◽  
Regulatory Mechanisms ◽  
Factor V ◽  
Factor Va ◽  
Dynamics Simulations ◽  
A2 Domain

Objective: The Australian snake ptFV ( Pseudonaja textilis venom-derived factor V) variant that retains cofactor function despite APC (activated protein C)-dependent proteolysis. Here, we aimed to unravel the mechanistic principles by determining the role of the absent Arg306 cleavage site that is required for the inactivation of Fva (mammalian factor Va). Approach and Results: Our findings show that in contrast to human FVa, APC-catalyzed proteolysis of ptFVa at Arg306 and Lys507 does not abrogate ptFVa cofactor function. Remarkably, the structural integrity of APC-proteolyzed ptFVa is maintained indicating that stable noncovalent interactions prevent A2-domain dissociation. Using Molecular Dynamics simulations, we uncovered key regions located in the A1 and A2 domain that may be at the basis of this remarkable characteristic. Conclusions: Taken together, we report a completely novel role for uniquely adapted regions in ptFVa that prevent A2 domain dissociation. As such, these results challenge our current understanding by which strict regulatory mechanisms control FVa activity.

scholarly journals A Prothrombinase-based Assay for Detection of Resistance to Activated Protein C

1996 ◽  
Vol 76 (03) ◽  
pp. 404-410 ◽  
Author(s):  
Gerry A F Nicolaes ◽  
M Christella ◽  
L G D Thomassen ◽  
Rene van Oerle ◽  
Karly Hamulyak ◽  
...  
Keyword(s):  
Protein C ◽  
Plasma Sample ◽  
Activated Protein C ◽  
Factor Xa ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Plasma Samples ◽  
Factor Va ◽  
Plasma Factor ◽  
Cofactor Activity

SummaryIn this paper we present a new method for the detection of resistance to activated protein C (APC) that is based on direct measurement of the effect of APC on the cofactor activity of plasma factor Va. The factor V present in a diluted plasma sample was activated with thrombin and its sensitivity towards APC was subsequently determined by incubation with phospholipids and APC. The loss of factor Va cofactor activity was quantified in a prothrombinase system containing purified prothrombin, factor Xa and phospholipid vesicles and using a chromogen-ic assay for quantitation of thrombin formation. The reaction conditions were optimized in order to distinguish normal, heterozygous and homozygous APC-resistant plasmas. Maximal differences in the response of these plasmas towards APC were observed when factor Va was inactivated by APC in the absence of protein S and when the cofactor activity of factor Va was determined at a low factor Xa concentration (0.3 nM).Addition of 0.2 nM APC and 20 μM phospholipid vesicles to a 1000-fold diluted sample of thrombin-activated normal plasma resulted in loss of more than 85% of the cofactor activity factor Va within 6 rnin. Under the same conditions, APC inactivated ∼ 60% and ∼20% of the factor Va present in plasma samples from APC-resistant individuals that were heterozygous or homozygous for the mutation Arg506⟶Gln in factor V, respectively. Discrimination between the plasma samples from normal and heterozygous and homozygous APC-resistant individuals was facilitated by introduction of the so-called APC-sensitivity ratio (APC-sr). The APC-sr was defined as the ratio of the factor Va cofactor activities determined in thrombin-activated plasma samples after 6 min incubation with or without 0.2 nM APC and was multiplied by 100 to obtain integers (APC-sr = {factor Va+APC/factor Va−APC} × 100). Clear differences were observed between the APC-sr of plasmas from normal healthy volunteers (APC-sr: 8-20, n = 33) and from individuals that were heterozygous (APC-sr: 35-50, n = 17) or homozygous APC resistant (APC-sr: 82-88, n = 7). There was no mutual overlap between the APC-sr of normal plasmas and plasmas from heterozygous or homozygous APC resistant individuals (p <0.0001). In all cases our test gave the same result as the DNA-based assay. Since the test is performed on a highly diluted plasma sample there is no interference by conditions that affect APC resistance tests that are based on clotting time determinations (e.g. coagulation factor deficiencies, oral anticoagulation, heparin treatment, the presence of lupus anticoagulants, pregnancy or the use of oral contraceptives). Furthermore, we show that part of the factor Va assay can be performed on an autoanalyzer which increases the number of plasma samples that can be handled simultaneously.

Protein C Is Responsible for the Rapid Inactivation of Factor Va following Blood Clotting In Vitro

1994 ◽  
Vol 72 (01) ◽  
pp. 070-073
Author(s):  
Denise E Jackson ◽  
Christina A Mitchell ◽  
Hatem H Salem
Keyword(s):  
Protein C ◽  
Blood Clotting ◽  
Essential Role ◽  
The Other ◽  
Factor V ◽  
Factor Va ◽  
Rapid Inactivation ◽  
Protein C Activity

SummaryWhen whole blood is allowed to clot in vitro, factor V is rapidly activated to factor Va which is subsequently inactivated. We developed two monoclonal anti-protein C antibodies, one of which inhibits protein C activation and the other inhibits protein C activity. The addition of either antibody to blood before clotting in vitro significantly inhibited the inactivation of factor Va, confirming the essential role of protein C in mediating the rapid inactivation of factor Va.

scholarly journals Functional role of the polysaccharide component of rabbit thrombomodulin proteoglycan. Effects on inactivation of thrombin by antithrombin, cleavage of fibrinogen by thrombin and thrombin-catalysed activation of factor V

1990 ◽  
Vol 270 (2) ◽  
pp. 419-425 ◽  
Author(s):  
M C Bourin ◽  
U Lindahl
Keyword(s):  
Protein C ◽  
Inhibitory Effect ◽  
Factor V ◽  
Promoting Effect ◽  
Activation Kinetics ◽  
Polysaccharide Chain ◽  
Factor Va ◽  
Kinetics Of ◽  
Similar Sample

Thrombomodulin (TM), a major anticoagulant protein at the vessel wall, serves as a potent cofactor for the activation of Protein C by thrombin. Previous work has indicated that (rabbit) TM is a proteoglycan that contains a single polysaccharide chain, tentatively identified as a sulphated galactosaminoglycan, and furthermore suggested that this component may be functionally related to additional anticoagulant activities expressed by the TM molecule [Bourin, Ohlin, Lane, Stenflo & Lindahl (1988) J. Biol. Chem. 263, 8044-8052]. Results of the present study establish that (enzymic) removal of the polysaccharide chain abolishes the inhibitory effect of TM on thrombin-induced fibrinogen clotting as well as the promoting effect of TM on the inactivation of thrombin by antithrombin, but does not affect the ability of TM to serve as a cofactor in the activation of Protein C. Studies of yet another biological activity of rabbit TM, namely the ability to prevent the activation of Factor V by thrombin [Esmon, Esmon & Harris (1982) J. Biol. Chem. 257, 7944-7947], confirmed that TM markedly delays the conversion of the native 330 kDa Factor V precursor into polypeptide intermediates, and further into the 96 kDa heavy chain and 71-74 kDa light-chain components of activated Factor Va. In contrast, the activation kinetics of a similar sample of Factor V incubated with thrombin in the presence of chondroitinase ABC-digested TM did not differ from that observed in the absence of TM. It is concluded that the inhibitory effect of TM on Factor V activation also depends on the presence of the polysaccharide component on the TM molecule.

Activated Protein C-Resistance Induced by a Low Molecular Weight Inhibitor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2136-2136
Author(s):  
Michael Kalafatis ◽  
Rihard E. Chiott ◽  
Richard F. Branda ◽  
Kenneth G. Mann
Keyword(s):  
Molecular Weight ◽  
Protein C ◽  
Activated Protein C ◽  
Low Molecular Weight ◽  
Factor V ◽  
Prolonged Incubation ◽  
Apc Resistance ◽  
Factor Va ◽  
Normal Plasma ◽  
Plasma Factor

Abstract Activated protein C (APC) inactivates factor Va (fVa) following three cleavages in the heavy chain at R506, R306 and R679. Cleavage at R506 precedes cleavage at R306. Cleavage at Arg306 is strictly lipid-dependent and results in total inactivation of the factor Va molecule with dissociation of fragments from the A2 domain from the rest of the molecule. Factor VLeiden is associated with an R→Q substitution at position 506 and is present at approximately 8% of the Caucasian population. The heterozygous presentation of factor VLeiden results in delayed inactivation of factor Va and “APC-resistance” with attendant increased risk of venous thrombosis. However, not all cases of “APC-resistance” are explained by factor VLeiden. We observed “APC-resistance” in a patient displaying heterozygous factor VLeiden, Waldenstrom’s macroglobulinenemia, systemic lupus erythrematosus (anticoagulant) and a history of coronary artery disease. The patient’s plasma resistance to APC inactivation was not repaired by immunodepletion of his factor VLeiden and replacement by normal plasma factor V. Conversely when the patient’s fVa was returned to factor V immunodepleted normal plasma it did not display APC-resistance. Cleavage of the patient’s plasma fVa at R306 was not detected following prolonged incubation of his clotted plasma at 37°C even when 2 nM APC was added following clotting. These data suggested that the APC-resistance observed in the patient was not due to the presence of factor VLeiden, but due to some property which inhibited the lipid dependent cleavage at Arg306. The patient’s plasma was depleted of IgG/IgM and the purified immunoglobulin fraction assessed for inhibition of APC cleavage and inactivation of fVa in a system using purified reagents. The data were compared with the inhibition of fVa inactivation by APC by an IgG/IgM fraction obtained from normal plasma under similar experimental conditions. No inhibition of APC cleavage and inactivation of fVa by the IgM/IgG fraction obtained from either plasma were observed. In addition, the fVa molecule contained in the IgM/IgG-depleted patient plasma was still resistant to cleavage and inactivation by APC. Following dialysis the patient’s plasma lost its ability to inhibit fVa cleavage and inactivation by APC. Overall these studies indicate that inhibition of fVa cleavage and inactivation by APC in the patient’s plasma is caused by a hitherto undescribed metabolite of low molecular weight. The mechanism of action of this metabolite is not yet known, but the evidence suggests that the metabolite interferes with the lipid-dependent cleavage and inactivation of fVa by APC at R306. These data demonstrate the existence of an as yet unknown APC inhibitor of low molecular weight in the plasma of a patient with lupus anticoagulant and severe thrombotic symptoms.

Differential Effect of An Autoantibody to Thrombin on Fibrinogen Cleavage and Protein C Activation.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3652-3652
Author(s):  
Aditi Shastri ◽  
Kelty R. Baker ◽  
Perumal Thiagarajan
Keyword(s):  
Protein C ◽  
Conflicts Of Interest ◽  
Joint Disease ◽  
Left Shoulder ◽  
Factor V ◽  
Dependent Manner ◽  
Thrombin Time ◽  
Bovine Thrombin ◽  
Normal Plasma ◽  
Human Thrombin

Abstract Abstract 3652 A 76 year old male experienced an unexpected blood loss of 1300 ml during an elective left shoulder replacement for degenerative joint disease. His post-operative course was uncomplicated. Further laboratory testing revealed a prolonged thrombin time of 33 seconds (control of 17.7–20.3 seconds). The reptilase time was within normal limits. He also had a normal factor V, × and fibrinogen level. The prolonged thrombin time failed to correct upon addition of normal plasma. The patient's IgG fraction was isolated by protein G-Sepharose chromatography and tested for its effect in coagulation assays. The patient's purified IgG (2.1 mg/ml) prolonged the thrombin time in normal plasma when tested with a commercial bovine thrombin time reagent. Under similar conditions, normal IgG had no effect. These laboratory tests suggest an acquired antibody to thrombin. The antibody was further characterized by testing its effect on purified human thrombin. The antibody prolonged the clotting time of human thrombin in normal plasma in a concentration dependent manner. However, the antibody had no effect on the esterolytic activity of human or bovine thrombin as measured by the proteolysis of the chromogenic thrombin substrate, S-2238. These results suggest that the epitope for the antibody overlaps the fibrinogen binding site on thrombin but does not include thrombin's active enzymatic site. We also tested the effect of the purified antibody on thrombomodulin-dependent thrombin activation of protein C. The patient's antibody was incubated with protein C, rabbit thrombomodulin and calcium containing buffer. The activation of protein C was measured using the chromogenic protein C substrate S2366. The antibody had no effect on protein C activation by thrombin. Acquired antibodies to thrombin develop after exposure to bovine thrombin, commonly used for surgical hemostasis. These antibodies are also associated with antibodies to factor V and a low factor V level. This patient's clinical and laboratory profile is intriguing, given the lack of previous exposure to bovine or human thrombin and the presence of normal Factor V levels. This novel antibody may represent formation of a spontaneous autoantibody to thrombin due to failure of immunological surveillance mechanisms. Disclosures: No relevant conflicts of interest to declare.

Different Anticoagulant Response to Activated Protein C (APC test) and to Agkistrodon Contortix Venom (ACV test) in a Family with FV-R506Q Substitution

1997 ◽  
Vol 3 (3) ◽  
pp. 168-173 ◽  
Author(s):  
Donate Gemrnati ◽  
Maria L. Serino ◽  
Rosella Mari ◽  
Isabella Verzola ◽  
Stefano Moratelli ◽  
...  
Keyword(s):  
Protein C ◽  
Dna Analysis ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Factor V ◽  
Heterozygous Condition ◽  
Apc Resistance ◽  
First Case ◽  
Plasma Factor ◽  
The Family

To identify the defect(s) responsible for the thrombotic condition affecting a 55-year-old male and his family, we have utilized a new methodological approach (ProC Global®, Istituto Behring, Milan, Italy) to screen the global anticoagulant activity of the protein C pathway, a defect that accounts for the majority of inherited thrombophilias. The test is based on the activation of endogenous protein C in plasma by Protac®, derived from Agkistrodon contortix snake venom (ACV test). Nineteen members of the family were investigated, 11 showed low responsiveness to ACV (normalized ACV ratios < 0.66; normal > 1. 12); in these individuals specific assays of protein C (PC) and protein S (PS) levels and normalized activated protein C ratios (n-APC-r) were performed. A second test evaluating response to APC, using the classic commercial APC test (n-APC-r 1), detected only 10 subjects with abnormal responses : the propositus and two members of the family with n-APC-r 1 values < 0.54, indicating the homozygous state for the R506Q factor V gene mutation, and seven with values ranging 0.69-0.83, consistent with the heterozygous condition (normal > 0.85). Although only ten subjects presented with low n-APC-r 1 values, DNA analysis, in agreement with the ACV test, detected 11 individual with factor V-R506Q substitution (two homozygotes and nine heterozygotes). Thus the classical APC test failed to identify the APC resistance phenotype in two heterozygous subjects whose values were clearly normal (1.05) in the first case and homozygous (0.53) in the second. The ACV test, however, and the modified APC test with test plasma 1/5 diluted in factor V-deficient plasma (n-APC-r 2) completely matched the DNA analysis. A phenotype/genotype correlation was observed in dilutions higher than 1/3 test plasma factor V-deficient plasma. The presence of unknown mechanisms that influence plasma response to exogenous preformed APC (normal at high factor V-deficient plasma dilutions) but not endogenous ACV activated PC was suspected. The suspected low levels of proteins C and S found in several R506Q members of the family were excluded by reassaying the anticoagulant activities at higher plasma dilution ; this supports the known influence of factor V Leiden on functional PC and PS clotting activity. We conclude that the ACV test is appropriate to evaluate the APC resistance condition, but for a firm diagnosis DNA analysis together with the modified APC test are strongly advised even in the presence of unquestionable APC-r values. Key Words: APC resistance-Factor V Leiden-APC test-ACV test-Diagnosis-Inherited thrombophilia.

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