Plasma Coagulability Research Articles

Effect of low or high doses of low‐molecular‐weight heparin on thrombin generation and other haemostasis parameters in critically ill patients with COVID‐19

The clinical picture of the coronavirus 2 (SARS-CoV-2)/COVID-19-related acute respiratory syndrome is often associated with a coagulopathy. An elevated D-dimer has been linked with an unfavorable prognosis in COVID-19 patients.

In Children with Provoked Venous Thromboembolism, Increasing Plasma Coagulability during the First 3 Months Postdiagnosis is Prognostic of Recurrence.

Prognostic factors for venous thromboembolism (VTE) recurrence following provoked VTE are largely unknown. Using the Clot Formation and Lysis (CloFAL) assay, single institutional research has shown overall improvement in acute hypercoagulability during the first 3 months postpediatric VTE, yet a rise in plasma coagulability in a subgroup of patients. We sought to define the incidence of rise in coagulability during the first 3 months post-provoked VTE, to investigate its relationship with elevated D-dimer, and to test the hypothesis that a marked rise in coagulability is independently prognostic of VTE recurrence. CloFAL and D-dimer assays were performed on plasma at 4 to 6 weeks and 3 months post-VTE in the Johns Hopkins pediatric VTE cohort and National Institutes of Health-sponsored Kids-DOTT trial. Associations of VTE recurrence with D-dimer and CloFAL assay measures were evaluated via logistic regression. Eighty-seven patients were included. Median follow-up was 1 year. Complete veno-occlusion was determined in 12% at 6 weeks. During the first 3 months post-VTE, a marked rise in coagulability was observed by CloFAL assay in 17% of patients, while D-dimer was elevated in 21%. Recurrent VTE occurred in 10% of patients. CloFAL assay, but not D-dimer, was associated with recurrence (odds ratio [OR] 5.87, 95% confidence interval [95% CI], 1.34-25.8]). After adjustment for veno-occlusion, patients with a marked rise in coagulability by CloFAL assay had a 10-fold increased risk of recurrent VTE (OR 10.33 [95% CI, 1.83-58.19]). Future work should seek to elucidate the mechanisms underlying a rise in plasma coagulability following provoked VTE and to substantiate its prognostic utility for recurrent VTE.

A Rise in Plasma Coagulability during the First 3 Months Following Provoked Venous Thromboembolism Is Associated with the Development of Post-Thrombotic Syndrome in Children and Young Adults: Analysis of the Kids-DOTT Multicenter Trial-Derived Biobank

Background:Knowledge of prognostic factors for the development of post-thrombotic syndrome (PTS) is limited, particularly in the pediatric population. Elevated plasma levels of D-dimer and FVIII, both markers of coagulation activation and inflammation, are associated with an increased risk of adverse thrombosis outcomes, including PTS, in children. Similarly, increased levels of inflammatory cytokines, such as interleukin-6 (IL-6), have been associated with venous endothelial dysfunction and fibrosis, suggesting a potential role in the development of PTS. Given the strong interplay between inflammation and coagulation, the aims of this study were to: (1)Investigate a hypothesized association between increased levels of plasma cytokines during the first 3 months post-diagnosis of deep venous thrombosis (DVT) with the development of PTS in patients <21 years old; and (2) to investigate an association between increased plasma coagulability during the first 3 months post-DVT diagnosis with the development of PTS. Methods:We used banked plasma biospecimens and masked clinical data from an ongoing NHLBI-sponsored multinational multicenter trial of VTE treatment in patients <21 years old (the Kids-DOTT trial, NCT00687882). All patients with an extremity DVT who underwent PTS assessment at 1 year were included in this analysis. The Clot Formation and Lysis (CloFAL) spectrophotometric assay was performed, as previously described, on banked plasma samples obtained at 6 weeks and 3 months post-VTE diagnosis. All samples were pre-treated with heparinase prior to assay. Assay measurements included maximal amplitude of the CloFAL waveform (MA), time to maximal amplitude (T1), and area under the curve at 60 minutes, indexed to that of the pooled normal plasma standard (AUC60). Levels of IL-6, IL-8, IL-12/IL-23p40, IL-17, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, interferon (IFN)-γ and C-reactive protein (CRP) were measured via V-Plex kit (Meso Scale Discovery, Rockville, MD). For each patient, the change in each laboratory parameter between the 3 months and 6 weeks post-VTE time points was calculated. The diagnosis of PTS was made at the one year follow-up assessment using the Manco-Johnson Instrument. Descriptive statistics were used to summarize data on patient and VTE characteristics as well as PTS outcome. Putative prognostic factors for PTS (age group, complete thrombus occlusion, CloFAL parameters, and inflammatory cytokines) were evaluated via univariate logistic regression, using odds ratios (OR) and 95% confidence intervals (CI). Variables with P-values <0.1 in univariate analyses were pre-specified for inclusion in an adjusted (multivariate) regression analysis. The blind was maintained in the Kids-DOTT trial throughout data transfer and analysis. Results: A total of 80 patients were included in the present analysis. Median age was 12.7 years (range 0.04 - 20.8 years). Patient and VTE characteristics at baseline, as well as PTS outcomes, are summarized in Table 1. PTS developed in 34% of patients by the 1 year post-VTE follow up visit. Univariate logistic regression analysis identified change in CloFAL AUC60 (OR=1.46, 95%CI=1.02-2.09; P=0.036) and complete thrombus occlusion at 6 weeks post-VTE (OR=3.29, 95%CI=0.93-11.6; P=0.064) as putative prognostic factors for PTS (Table 2). After adjustment in multivariate regression, change in CloFAL AUC60 remained the only possibly-significant (P=0.06) independent prognostic factor for PTS. Specifically, for each 1 A.U. increase in CloFAL AUC from the 6 weeks to 3 months post-VTE diagnosis, the odds of PTS increased by more than 40% (OR=1.43, 95%CI=0.99-2.08; Table 3). Changes in none of the measured inflammatory cytokines were prognostic of PTS. Conclusions: PTS developed in 34% of patients <21 years old enrolled in enrolled in an ongoing multicenter trial of provoked DVT treatment. A rise in plasma coagulability during the first 3 months post-DVT diagnosis, as measured by increase in AUC60 in the CloFAL assay is a candidate prognostic factor for PTS in patients <21 years old with provoked DVT. Future work should seek to investigate the mechanisms of increased plasma coagulability following a course of anticoagulation therapy in young VTE patients, and to substantiate findings of its potential prognostic significance for the development of PTS. Disclosures Everett: ImmunArray: Consultancy, Patents & Royalties. Goldenberg:NIH: Other: research support and salary support.

Thrombodynamic parameters of hypercoagulation of blood in children with childhood autism and schizophrenia

To detect blood plasma coagulability in children with mental diseases using a thrombodynamics test. The study included two groups of children. Group 1 included 11 patients with infantile psychosis in autism (F84.02) (4 girls and 7 boys). Group 2 included 8 patients with childhood schizophrenia (F20.8xx3) (4 girls and 14 boys). A test was performed with T-2 Thrombodynamics analyzer (LLC Hemacore, Moscow, Russia). Thrombodynamic parameters, such as initial, steady-state velocity and spontaneous clots adjusted velocity (Vi, Vst and V, μm/min, respectively) and clot size at 30 minute of thrombodynamics test (CS μm) were significantly increased in the total group of patients (n=29). The time of appearance of spontaneous clots (Tsp), the time of clot lag time (Tlag) and clot density (D) did not differ significantly from the normal values (p=0.98; p=0.27 and p=0.21, respectively). In the autism group (n=11), Vi, Vst and V were significantly higher than normal values, while CS, Tsp, Tlag and D did not differ from norm. In the schizophrenia group (n=18) V, Vst and CS, and Vi were significantly increased. Tsp, Tlag and D did not differ from normal values. Differences between the parameters of thrombodynamics in 1 and 2 groups were not statistical significant. It was shown for the first time that clotting (hypercoagulability) of the blood plasma in patients with autism and childhood schizophrenia was increased. This can cause thrombosis in small vessels of the brain. Early spontaneous clots appear in many patients that indicating the presence of systemic inflammation, possibly associated with an exacerbation of neuroinflammation. The thrombodynamics test allows detection of predisposition to hypercoagulability in the early stages when other methods are not sensitive enough.

Evaluation of Blood Plasma Coagulability by Laser Speckle Correlation

The process of blood plasma coagulation was studied using the laser speckle correlation technique. Coagulation times measured in samples of different volumes using the Minilab 701 commercial coagulometer and the laser speckle correlation technique were compared. It was shown that the laser speckle correlation technique provides comparable accuracy of measurement, while allowing the sample volume to be considerably reduced.

Plasma Serum Amyloid A Levels Are Increased In Venous Thrombosis Patients and Are Correlated with Blood Coagulability

AbstractAbstract 155Inflammation is well correlated with cardiovascular diseases, but its relationship to venous thromboembolic disease (VTE) is less well established. One well studied inflammatory marker, C-reactive protein (CRP), has been inconsistently linked to VTE and this association is controversial. First, we explored the relationship between serum amyloid A (SAA), an inflammatory biomarker, and VTE because SAA can induce tissue factor (TF) expression and release pro-inflammatory cytokines, activities that might imply a linkage between SAA elevation and thrombotic disease. Second, we probed the influence of SAA on blood coagulability. Plasma SAA levels were determined by ELISA for Scripps VTE registry subjects (113 VTE patients and 113 matched controls). Inclusion criteria for this study included age at thrombosis < 55 years, > 3 months since diagnosis of VTE, a life expectancy of > 3 years, no cancer, and no lipid lowering medications. VTE patients had significantly higher median levels of SAA than controls (3.70 vs. 2.12 μ g/ml, p<0.0001). Odds ratio (OR) calculation based on SAA values > 90th percentile of controls showed that elevated SAA was associated with VTE with an OR value of 6.9 (95% CI: 3.3–14). Since SAA is associated with other inflammation parameters, we measured two other acute-phase inflammatory proteins. SAA was correlated with CRP and fibrinogen (r=0.32, p<0.0001 and r=0.37, p<0.0001, respectively). To test if the association of elevated plasma SAA levels with VTE was primary or, alternatively, only secondary to the association of SAA with other acute-phase proteins (e.g., CRP or fibrinogen), we adjusted the OR value (SAA > 90th percentile of controls) for CRP and fibrinogen. However, even after this adjustment for these two inflammation biomarkers, the OR for association of SAA with VTE was not altered (adjusted OR=6.8, 95%CI: 3.1–15). Thus, elevated plasma SAA (> 90th percentile of controls) is a strikingly strong biomarker for VTE that appears to be independent of two other well studied inflammatory biomarkers. To assess the potential direct relationship between SAA in plasma and blood coagulation reactions, we obtained data using endogenous thrombin potential assays and other coagulation assays. Plasma SAA levels from individual donors were very well correlated with parameters of TF-induced thrombin generation in plasma, showing that endogenous plasma SAA levels were associated with plasma coagulability. When added to plasma, purified recombinant SAA dose-dependently enhanced TF-induced thrombin generation. This procoagulant activity of endogenously added SAA that was seen in TF-induced thrombin generation assays was not affected by corn trypsin inhibitor, a specific inhibitor of factor XIIa, implying that contact activation was not mediating this SAA procoagulant activity. However, the procoagulant activity of purified recombinant SAA added to plasma was not seen when factor XI deficient plasma was used for assays, suggesting that SAA exerts procoagulant effects in plasma that require factor XI, i.e., effects that involve factor XI activation and/or expression of factor XIa activity. Further studies are needed to clarify details for the procoagulant effects of SAA on coagulation pathways. In conclusion, these results show that elevated plasma levels of SAA are strongly linked to VTE in adults (< 55 yrs old) and imply that SAA itself is a potential enhancer of thrombin generation in plasma acting via factor XI and the intrinsic coagulation pathway. Disclosures:No relevant conflicts of interest to declare.

Evidence of Increased Plasma Coagulative Capacity by CloFAL Assay among Pediatric Factor V Leiden and Prothrombin G20210A Heterozygotes with, Versus without, a First-or Second-Degree Family History of Venous Thromboembolism

BACKGROUND: The factor V (FV) Leiden and prothrombin (PT) G20210A polymorphisms in heterozygous state are present in 5% and 1–2% of Caucasians, respectively, and confer approximately 5-fold and 2-fold increases in the risk of incident venous thromboembolism (VTE). While some families who carry these genetic thrombophilia traits exhibit a prothrombotic phenotype, others have no (or only a limited) history of VTE. The ability to discern which individuals with personal and familial carriage of these genetic thrombophilias possess a clinically meaningful increase in VTE risk remains elusive, and (particularly among children) is perhaps best informed presently by family history of VTE.OBJECTIVE AND HYPOTHESES: We sought to evaluate overall plasma coagulative capacity in FV Leiden and PT G20210A heterozygotes using the Clot Formation and Lysis (CloFAL) assay, a global turbidimetric plasma assay of tissue-factor induced fibrin clot formation and tissue-type plasminogen activator enhanced fibrinolysis. We hypothesized that children heterozygous for either thrombophilia would not uniformly demonstrate hypercoagulability, but that coagulative capacity would be increased among heterozygotes who have a family history of VTE.PATIENTS AND METHODS: Children aged birth to 18 years (inclusive) enrolled in prospective inceptional cohort study of thrombosis/thrombophilia/stroke were included in the analysis if they were found to be heterozygous for FV Leiden or PT G20210A upon comprehensive thrombophilia testing and had undergone CloFAL assay testing on a research basis. Data on personal and family history of thrombotic events, thrombophilia testing, and CloFAL assay findings were analyzed. Intergroup comparisons of continuous data were performed by Mann-Whitney U test and proportions were compared between groups using chi-square or Fisher's exact test, as appropriate.RESULTS: Characteristics of the study population are shown in Table 1. Approximately 70% of patients were evaluated for a family history of VTE (with/without known thrombophilia) and nearly 50% had personal histories of VTE or arterial ischemic stroke (AIS)/recurrent transient ischemic attack (TIA); those evaluated for events were significantly older than those without events, and this difference was statistically significant among those with a positive family history fo VTE. Hypercoagulability was shown in 50% of patients and hypofibrinolysis in 13% using the CloFAL assay. Plasma coagulative capacity and maximal amplitude (MA) of the CloFAL waveform were significantly increased in patients with, versus without, family history of VTE (coagulation index, CI: 102% vs. 72% of the adult normal pooled plasma standard, respectively, p=0.04; MA: 0.415 vs. 0.322, p=0.02), and were not explained by age differences between groups. However, in this relatively small study population, the proportion of CloFAL CI results that exceeded the upper limit of normal values did not significantly differ between those with, versus without, family history of VTE. Pediatric FV Leiden or PT G20210A heterozygotes with positive family history of VTE were more likely to have multi-trait (>1) thrombophilia, in which case a trend toward increased plasma coagulability was demonstrated (CI: 139% [multi-trait] vs. 86% [isolated trait]; p=0.07); superimposed thrombophilias in this group most often consisted of elevated factor VIII activity and Lp(a) concentration.CONCLUSIONS: The present findings using the CloFAL global assay indicate that, while pediatric FV Leiden or PT G20210A heterozygotes do not uniformly exhibit hypercoagulability, plasma coagulative capacity is nevertheless significantly increased among heterozgyotes who have a family history of VTE, which may relate to the presence of superimposed thrombophilias.Table 1. Summary characteristics of the study population.N32Median age at evaluation (range)9.5 y (1–18 y)Personal history of events*14 y (1–18 y)No personal history of events*8 y (2–18 y)FV Leiden heterozygote (n)26**PT G20210A heterozygote (n)8**Personal history of VTE (n)11Personal history of AIS/recurrent TIA4Family history (1st/2nd degree) of VTE71%Multi-trait (>1) thrombophilia45%Acquired thrombophilia24%Hypercoagulability by CloFAL assay50%Hypofibrinolysis by CloFAL assay13%*VTE, AIS, or recurrent TIA**Two patients were dual heterozygotes.

Influence of factor IX on overall plasma coagulability and fibrinolytic potential as measured by global assay: monitoring in haemophilia B

We sought to determine the influence of factor IX (FIX) deficiency upon overall coagulative and fibrinolytic capacities in plasma using the clot formation and lysis (CloFAL) assay, and to investigate the role of this global assay as an adjunctive monitoring tool in haemophilia B. CloFAL assay parameters were measured in vitro in platelet-poor plasma in relation to FIX activity and antigen (FIX:Ag), and were determined ex vivo among FIX-deficient patients (n = 41) in comparison to healthy individuals (n = 48). Supplementation of FIX-deficient plasma with FIX in vitro demonstrated a non-linear concentration dependence of FIX upon overall plasma coagulability. Ex vivo, coagulability was significantly decreased in FIX-deficient vs. healthy subjects among adults [median coagulation index (CI): 4% vs. 104% respectively; P < 0.001] and children (median CI: 9% vs. 63%; P < 0.001). Fibrinolytic capacity was increased in adult FIX-deficient vs. healthy subjects (median fibrinolytic index: 216% vs. 125%, respectively, P < 0.001), and was supported by a trend in shortened euglobulin lysis time (ELT). Severe haemophilia B patients showed heterogeneity in aberrant CloFAL assay waveforms, influenced partly by FIX:Ag levels. Patients with relatively preserved FIX:Ag (i.e. dysfunctional FIX) exhibited a shorter time to maximal amplitude in clot formation than those with type I deficiency. During patient treatment monitoring, markedly hypocoagulable CloFAL assay waveforms normalized following 100% correction with infused FIX. The CloFAL global assay detects FIX deficiency, demonstrates differences in coagulability between dysfunctional FIX and type I deficiency, and appears useful as an adjunctive test to routine FIX measurement in monitoring haemophilia B treatment.

Influence of Factor IX on Overall Plasma Coagulability and Fibrinolytic Potential in Hemophilia B as Measured by Global Assay.

BACKGROUND: The Clot Formation and Lysis (CloFAL) global assay is a turbidimetric plasma assay utilizing coagluation activation by lipidated tissue factor and fibrinolytic enhancement by tissue plasminogen activator, and has recently been shown to be sensitive for factor VIII (FVIII) deficiency (Goldenberg et al., Thromb Res 2005; Goldenberg et al., Haemophilia 2006, in press). Deficiency of factor IX (FIX) is often clinically less severe than that of FVIII. We sought to evaluate the analytical sensitivity of the CloFAL assay for FIX deficiency, both in vitro and ex vivo.METHODS: The influence of FIX activity upon the CloFAL assay was examined in vitro in FIX-deficient plasma by mixing study with pooled normal plasma, addition of plasma-derived FIX concentrate, and repletion with recombinant FIX. The analytical sensitivity of the CloFAL assay for FIX deficiency was then studied clinically via comparison of healthy individuals to FIX-deficient adults (n=25) and children (n=19), of whom greater than 1/3 had mild hemophilia B (FIX activity >/= 5.0 U/dL). FIX-deficient subjects had not received exogenous FIX within the prior 96 hours and had no evidence of active hepatitis. Healthy adult and pediatric groups (n=25 each) were without chronic illness or family history of bleeding or thrombosis. None of the study subjects were taking medications that affect hemostasis or had recent active bleeding or acute infection.RESULTS: Alteration of FIX activity in vitro exerted considerable influence upon the CloFAL assay (Figure 1). Ex vivo, the CloFAL assay coagulation index (CI), a measure of the area under the clotting curve, was significantly decreased in FIX-deficient versus healthy subjects among adults and children alike (median CI, adults: 10% vs. 94%, respectively; median CI, children: 10% vs. 63%, respectively; P < 0.0001 for each), and correlated significantly with FIX activity in both age groups (r = 0.80 and r = 0.77; P < 0.0001 for each). In addition, the CloFAL assay fibrinolytic index (FI) was increased in FIX-deficient versus healthy subjects (median FI, adults: 228% vs. 109%, respectively, p<0.0001; median FI, children: 276% vs. 202%, respectively; p=0.7), although this difference was not statistically significant in children. Interestingly, severe hemophilia B patients (n=8; FIX activity < 1.0 U/dL) showed considerable heterogeneity in CloFAL assay waveforms (Figure 2). Nevertheless, the assay uniformly discriminated these patients from healthy controls.CONLUSION: This work demonstrates that the CloFAL global assay, which is highly influenced by FVIII activity, is also analytically sensitive to FIX deficiency. [Display omitted] [Display omitted]

Femoral head blood flow reduction and hypercoagulability under 24 h megadose steroid treatment in pigs

The pathogenesis of corticosteroid-induced femoral head necrosis is assumed to be ischemia. The purpose of this study was to investigate the perfusion pattern of the femoral head and plasma coagulability during 24 h corticosteroid megadose treatment, as recommended by the National Acute Spinal Cord Injury Studies (NASCIS), in the awake big animal model. Blindedly, 9 animals underwent megadose methylprednisolone infusion (30 mg/kg intravenously as an initial bolus, followed by 5.4 mg/kg/h for further 23 h) while 9 animals served as placebo treated controls. Regional blood flow of the systematically subdivided femoral head, proximal femur, acetabulum, and soft tissue hip regions was investigated by the microsphere technique at steady state (phase 1), after the initial bolus infusion (phase 2), and after the completed treatment (phase 3). Plasma coagulability was examined in phases 1 and 3. Blood flow of the femoral head epiphysis and metaphyseal cancellous bone was unchanged after one hour of steroid infusion, but decreased after the completed treatment at 24 h in the experimental group. Femoral head blood flow reduction was global without a tendency to more pronounced blood flow decrease in any subregion. Plasma fibrinogen was significantly higher after 24 h of steroid infusion than in the placebo control group. 24 h high dose methylprednisolone treatment causes femoral head blood flow reduction and hypercoagulability of plasma in the normal awake immature pig. These findings may be pathogenetic factors in the early stage of steroid-induced osteonecrosis.

Platelet activation and hypercoagulability following treatment with porcine factor VIII (HYATE:C).

Activation of platelets and coagulation in vivo was studied in nine patients with hemophilia A and inhibitors to human Factor VIII, prior to and following treatment with porcine Factor VIII (PFVIII; HYATE:C). In addition, six hemophiliac patients were similarly studied after treatment with recombinant Factor VIII (rFVIII). Platelet activation was also examined in vitro using porcine von Willebrand factor (PvWF)-enriched and PvWF-depleted fractions obtained by fractionation of PFVIII. Coagulation was assessed by measuring the concentrations of plasma prothrombin fragment 1+2 concentrations (prothrombinase generation) and Factor Xa-ATIII. Patients treated with PFVIII had significantly increased numbers of circulating platelets expressing CD62 and CD63 (markers of platelet activation) and annexin V (marker of platelet procoagulant activity) compared to patients treated with rFVIII; the former patients also demonstrated an increase in plasma coagulability after therapy. In in vitro experiments it was observed that the platelet-activating and procoagulant capacity of PFVIII resided in the PvWF-enriched fraction, and the same was true for the plasma hypercoagulability following exposure of platelets to PFVIII. These results support the hypothesis that PFVIII-induced platelet activation provides a mechanism for enhancing hemostasis, separate from, and additional to, that due to increased circulating Factor VIII, and it is due to residual PvWF in the PFVIII preparation.

Virucidal heat-treatment of single plasma units: a potential approach for developing countries.

Since HIV first burst onto the scene of transfusion medicine, the quest for viral inactivation processes for plasma and plasma products has not ceased. Sophisticated methods for improving viral safety are currently used in the industrial world. However, in developing countries, with no facilities for treating plasma, nonviral-inactivated fresh frozen plasma [FFP] continues to be used extensively, and as screening may not be optimal (or may even be absent), FFP still contributes to the spread of HIV and other infectious viruses. The feasibility of heat-treating FFP at the liquid state, in its collection bag, was explored by testing diverse conditions of temperature and duration, in the presence of biologically compatible stabilisers. Quality of the heat-treated plasma was evaluated by haematological, biochemical and animal assays. The efficiency of the method to inactivate viruses was validated using HIV and model viruses. The selected heating conditions are 50 degrees C for 3 h. The optimized combination of stabilizers is composed of 30 mM trisodium citrate, 10 g L-1 L-lysine, 12 mM calcium gluconate and 150 g L-1 sorbitol. Plasma coagulability is appropriately preserved as shown by the KCT ratio (1.4). Recovery of biological activity of most coagulation factors is higher than 70% (including fibrinogen & von Willebrand factor). Electrophoretic and immunoblotting studies did not evidence protein aggregation and/or degradation. Viral validation studies of this procedure have shown complete inactivation of HIV (> 6.6 log) in less than 1 h of treatment. A viral reduction of at least 4 log for various model viruses, including those of hepatitis A and C viruses, suggests a potential contribution of the method to diminish the risk from various blood-borne viruses. The selected formulation appears to preserve plasma protein integrity and properties. The procedure does not require sophisticated equipment but it is mandatory to monitor it carefully to ensure quality and reproducibility. If properly controlled and standardized, this approach offers an opportunity to reduce the risk of transmission of HIV and other viruses, particularly in poor countries with a high incidence of HIV.

Fibrin(ogen) degradation by a 24k-endopeptidase from a Chryseobacterium Sp.

Summary A novel 24k-endopeptidase purified from the conditioned medium of a novel species of the Chryseobacterium genus, degraded purified fibrinogen and fibrin. At an enzyme/substrate ratio of 1/200 to 1/1000 rapid degradation of the human fibrinogen α-chain occurred and delayed degradation of the β-chain, whereas the γ-chain was more resistant. Degradation was associated with loss of thrombin-inducible coagulation. Incubation of 24k-endopeptidase (500 nM) in human plasma at 37°C for 3 h resulted in a decrease in fibrinogen to 38 ± 7% of baseline, and an increase of the thrombin time from 21 ± 1 to 52 ± 17 s. Other coagulation factors as well as plasminogen were not significantly affected. Fibrinogen digested with 24k-endopeptidase did not confer anticoagulant properties when added to human plasma.In a buffer milieu,125I-fibrin labelled matrix was lysed in a time- and concentration-dependent manner by 24k-endopeptidase. Purified125I-labelled fibrin clots (65 μl) in a buffer milieu (500 μl) were also efficiently lysed, requiring 50 nM 24k-endopeptidase for 50% lysis in 1 h. The presence of a physiological fibrinogen concentration in this assay did not affect lysis. In contrast, no significant lysis of125I-fibrin labelled plasma clots (65 μl) in human plasma (500 μl) was observed upon incubation with 24k-endopeptidase (up to 1 μM) for up to 24 h. Incubation of 24k-endopeptidase with α2-macroglobulin resulted in formation of a complex with impaired proteolytic activity against fibrinogen. These findings indicate that 24k-endopeptidase may reduce plasma coagulability by degradation of fibrinogen and that its differential action on fibrin in a buffer milieu or in plasma may be due to inhibition by α2-macroglobulin.

Effect of an herbal protein, CI-1, purified fromCajanus indicus, in models of liver failure in mice

Acute liver damage models in Swiss albino mice (male; 25–30 g) induced by paracetamol (300–500 mg/kg); β-galactosamine (500 mg/kg); and 40% ethanol (2 ml / 100 g) were studied. Clinical indications of chronic hepatocellular necrosis were manifested within 24 h of toxic doses. Initially, serum transaminases, alkaline phosphatase, lactate dehydrogenase, bilirubin, and triglycerides were markedly increased; in addition, the plasma prothrombin time was prolonged. The total serum protein and serum albumin were significantly decreased in comparison to controls. Treatment of severely liver-injured mice with a protein, CI-1, purified from the leaves of Cajanus indicus at a dose of 100 μg/ml i.p. regularly for 14 days significantly lowered the respective serum enzymes such as transaminases (SGOT, SGPT), alkaline phosphatases (ALK), and lactate dehydrogenase (LDH), prevented the loss of liver protein content, and improved the altered plasma coagulability. Histological studies of liver of treated (hepatoxins) mice reveal massive centrilobular necrosis, Kupffer's cells hyperplasia, and periportal fatty changes in contrast to control animals. Treatment with CI-1 in liver-damaged animals showed near normal histology. CI-1 may be a useful approach in the treatment of liver disorders. Drug Dev. Res. 48:76–83, 1999. © 1999 Wiley-Liss, Inc.

Migraine and prothrombotic genetic risk factors.

It has been suggested that during attacks of migraine both platelet activation and plasma coagulability are increased. We investigated the prevalence of several prothrombotic genetic risk factors in patients with migraine: factor V R/Q 506, factor II 20210 G/A, decanucleotide insertion/deletion in the factor VII promoter, and the platelet HPA-1 and HPA-2 alloantigenic systems, by genotypic identification in an age- and sex-matched case-control study including 106 patients with migraine (49 with aura, and 57 without aura). The prevalence of all genotypes was similar among case patients and controls. No association in relation to the type of migraine was detected in the factor II, factor VII, HPA-1, or HPA-2 polymorphisms. Our results showed a high prevalence of factor V Leiden in those patients with migraine with aura (6.1%), though that association was not statistically significant. The studied prothrombotic genetic factors do not seem to be associated with the development of migraine and, therefore, are not likely relevant in the previously reported hypercoagulability and platelet hyperaggregability in this disease.

Migraine and prothrombotic genetic risk factors.

It has been suggested that during attacks of migraine both platelet activation and plasma coagulability are increased. We investigated the prevalence of several prothrombotic genetic risk factors in patients with migraine: factor V R/Q 506, factor II 20210 G/A, decanucleotide insertion/deletion in the factor VII promoter, and the platelet HPA-1 and HPA-2 alloantigenic systems, by genotypic identification in an age- and sex-matched case-control study including 106 patients with migraine (49 with aura, and 57 without aura). The prevalence of all genotypes was similar among case patients and controls. No association in relation to the type of migraine was detected in the factor II, factor VII, HPA-1, or HPA-2 polymorphisms. Our results showed a high prevalence of factor V Leiden in those patients with migraine with aura (6.1%), though that association was not statistically significant. The studied prothrombotic genetic factors do not seem to be associated with the development of migraine and, therefore, are not likely relevant in the previously reported hypercoagulability and platelet hyperaggregability in this disease.

Influence of heparin, protamine and polybrene on the time integral of thrombin generation (endogenous thrombin potential).

We investigated the anticoagulating and heparin-neutralizing properties of protamine and polybrene (hexadimethrine bromide), using the endogenous thrombin potential (ETP) as the parameter to access plasma coagulability. The hypocoagulability induced by high doses of heparin (3 IU/ml) could be reversed by addition of protamine to a very limited extent only. Polybrene on the other hand did neutralize heparin at the equivalent concentration and a two-fold excess did not influence the ETP parameters. In vivo neutralization of high-dose heparin with protamine should therefore be reconsidered. Our experiments suggest polybrene to be superior over protamine with respect to neutralization of high doses of heparin.

Platelet - vessel wall interaction: role of blood clotting.

Vascular damage initiates not only the adhesion and aggregation of blood platelets but also coagulation, which is of mixed (intrinsic and extrinsic) origin. Evidence is presented that thrombin, generated as a result of the injury, is a prerequisite for platelet aggregation. Platelets, after activation, in their turn promote coagulation. Prostaglandin I2 (PGI2 or prostacyclin) inhibits coagulation induced by damaged vascular tissue. This effect of PGI2 is mediated by the inhibition of platelets in their participation in the generation of factor Xa and thrombin. Dietary cod liver oil, by changing plasma coagulability, decreases the procoagulation activity of vessel walls, and arterial thrombosis. Another fish oil with similar effects on plasma coagulability and some other haemostatic parameters does not modify vessel wall-induced clotting, nor does it significantly lower arterial thrombosis tendency; this indicates the physiological relevance of vessel wall-induced clotting in arterial thrombus formation. Some evidence is also given for the importance of vessel wall-induced clotting in primary haemostasis.

Changes in blood clotting systems during migraine attacks.

SYNOPSISThe activity of various blood clotting systems was measured during attacks of migraine complicated by prolonged focal cerebral dysfunction and contrasted with those observed when the same and other patients had uncomplicated migraine. Results differed in the two groups.In patients with complicated migraine, plasma coagulability rose during both complicated and uncomplicated attacks. During a complicated migraine attack, platelet aggregability to ADP also rose in all but one patient. Platelet aggregability fell in the same patients when they had uncomplicated migraine. Plasma coagulability consistently increased more when the headache was associated with focal cerebral dysfunction.In patients with uncomplicated migraine, plasma coagulability dropped during the later phase of their attacks. This finding may reflect the release of endogenous heparin from mast cells and basophilic leucocytes. Platelet aggregability to ADP decreased in some but rose in others.The results support the hypothesis that prolonged focal cerebral dysfunction may reflect thromboembolic phenomena. The influence of estrogens and genetic factors on clotting systems is reviewed.

Effect of beta-sitosterol on regression of hyperlipemia and increased plasma coagulability in the chicken.

Summary1. A cholesterol-induced hyperlipemia was fully counteracted (reduced to normal levels) by feeding beta-sitosterol.2. The decreased plasma clotting times associated with a hyperlipemia returned to control values when a normal lipid level was attained.3. An inverse relationship was demonstrated between plasma clotting time in presence of Russell's viper venom and degree of lipemia; i.e., a hyperlipemia resulted in decreased plasma clotting time, and vice versa. 4. Addition of 4% beta-sitosterol to the diet prevented or reversed an increased lipid and cholesterol concentration in liver and aorta.