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J H Griffin

Publications and source records attributed to J H Griffin.

At least 19 recordsLinked to original sources
PubMed2006

Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and improved potency in whole blood clotting assays.

BACKGROUND: Genetically engineered disulfide bonds in B-domain-deleted factor (F) VIII variants (C662-C1828 FVIII and C664-C1826 FVIII) improve FVIIIa stability by blocking A2 domain dissociation because the new disulfide covalently links the A2 and A3 domains in FVIIIa. AIM: The aim of this study was to assess the hypothesis that these variants have physiologically relevant properties because of prolonged thrombin generation and improved clot formation in whole blood. METHODS: Clot-formation properties in whole blood were measured in thromboelastogram assays. The thrombin generation capabilities of the wild-type (WT) FVIII and FVIII variants were determined, and half-lives of FVIIIa variants were determined in fresh whole blood serum. RESULTS: Thromboelastogram assays were performed with fresh, severe hemophilia whole blood reconstituted with variant and WT FVIII. The two disulfide bond-stabilized FVIII variants and WT FVIII had comparable clotting times at all studied concentrations. However, when compared with WT FVIII at low concentrations, the two FVIII variants required only 10% as much FVIII to achieve comparable clot-formation rates, clot-formation times and clot firmness values. The differences between WT and FVIII variants were quite pronounced at low FVIII concentrations. Measurement of the endogenous thrombin potential in FVIII-deficient plasma supplemented with these FVIII variants confirmed that the disulfide bond-stabilized variants supported high levels of thrombin generation at lower concentrations than did WT FVIII. During the course of clot generation in whole blood, the disulfide bond-stabilized FVIIIa variants had approximately 5-fold increased half-lives relative to WT FVIIIa. CONCLUSION: C662-C1828 FVIII and C664-C1826 FVIII have physiologically relevant superior clot-forming properties in a whole blood environment, most likely due to the increased half-life of these FVIIIa variants in whole blood.

Blood Coagulation↗
PubMed2006

Limited generation of activated protein C during infusion of the protein C activator thrombin analog W215A/E217A in primates.

Anticoagulation with activated protein C (APC) reduces the mortality of severe sepsis. We investigated whether the circulating protein C (PC) pool could be utilized for sustained anticoagulation by endogenous APC. To generate APC without procoagulant effects, we administered the anticoagulant thrombin mutant W215A;E217A (WE) to baboons. In preliminary studies, administration of high dose WE (110 microg kg(-1) i.v. bolus every 120 min; n = 2) depleted PC levels by 50% and elicited transient APC bursts and anticoagulation. The response to WE became smaller with each successive injection. Low dose WE infusion (5 microg kg(-1) loading + 5 microg kg(-1) h(-1) infusion; n = 5) decreased plasma PC activity by 15%, from 105% to 90%, to a new equilibrium within 60 min. APC levels increased from 7.5 ng mL(-1) to 86 ng mL(-1) by 40 min, then declined, but remained elevated at 34 ng mL(-1) at 240 min. A 22-fold higher dose WE (n = 5) decreased PC levels to 60% by 60 min without significant further depletion in 5 h. The APC level was 201 ng mL(-1) at 40 min and decreased to 20 ng mL(-1) within 120 min despite continued activator infusion. Co-infusion of WE and equimolar soluble thrombomodulin (n = 5) rapidly consumed about 80% of the PC pool with significant temporal increase in APC generation. In conclusion, low-grade PC activation by WE produced sustained, clinically relevant levels of circulating APC. Limited PC consumption in WE excess was consistent with the rapid depletion of cofactor activity before depletion of the PC zymogen. Reduced utilization of circulating PC might have similar mechanism in some patients.

Amino Acid Substitution↗
PubMed2006

Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants.

BACKGROUND: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. AIMS: Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII). METHODS: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. RESULTS: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. CONCLUSION: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.

Factor IXa↗
PubMed2005

Cholesterol enhances phospholipid-dependent activated protein C anticoagulant activity.

The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities.

Anticoagulants↗
PubMed2005

Decreased plasma sensitivity to activated protein C by oral contraceptives is associated with decreases in plasma glucosylceramide.

Oral contraceptive (OC) use increases venous thrombosis (VTE) risk and causes activated protein C (APC) resistance. Plasma glucosylceramide (GlcCer) deficiency is associated with VTE and GlcCer functions as an APC anticoagulant cofactor. Because estradiol decreases GlcCer in cultured cells, we hypothesized OC use would decrease plasma GlcCer and contribute to APC resistance. In a pilot study, seven female adults alternatively took second and third generation OCs and plasma samples were analyzed for GlcCer using high performance liquid chromatography and for APC sensitivity using modified prothrombin time assays. Second and third generation OC usage decreased the APC sensitivity ratio by 8.1% +/- 4.7% (P = 0.004) and 11.7% +/- 8.2% (P = 0.013) and plasma GlcCer levels by 10.1% +/- 6.8% (P = 0.008) and 11.0% +/- 5.1% (P = 0.002), respectively. The plasma GlcCer level correlated with the sensitivity of plasma to APC (P = 0.017, r = 0.51, n = 21 plasma samples). Thus, both second and third generation OC usage decreased plasma GlcCer which could cause a reduction in the plasma sensitivity to APC/protein S, thereby potentially increasing VTE risk.

Activated Protein C Resistance↗
PubMed2004

Thrombophilic factors are not the leading cause of thrombosis in Behçet's disease.

BACKGROUND: Venous and arterial thromboses occur in patients with Behçet's disease and are associated with significant morbidity and mortality. Studies on a possible association between the occurrence of thrombosis and thrombophilia in patients with this disease have been controversial. OBJECTIVE: To determine the prevalence of the most common thrombophilias and dyslipidaemia in patients with Behçet's disease with and without thrombosis. METHODS: Blood samples from 107 patients with Behçet's disease who had or did not have thrombosis were analysed for factor V Leiden, prothrombin G20210A polymorphism, methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, factor VIII level, homocysteine and C reactive protein concentrations, dyslipidaemia, and plasma glucosylceramide. RESULTS: There was no difference between patients with and without thrombosis in the prevalence of prothrombin G20210A polymorphism, factor V Leiden, homozygous MTHFR C677T, or plasma concentrations of homocysteine, C reactive protein, or glucosylceramide. In contrast, patients with thrombosis were found to have significantly higher mean levels of factor VIII, total cholesterol, triglycerides, VLDL cholesterol, and apolipoproteins B-100, C-II, and C-III than those without thrombosis. Multistepwise logistic regression analysis showed that triglyceride concentration was the best marker associated with thrombosis (p = 0.008), with an estimated odds ratio of 1.58 (95% confidence interval, 1.09 to 2.30) for a difference of 40 mg/dl. CONCLUSIONS: Thrombophilia does not seem to play a major role in the tendency to thrombosis in Behçet's disease. However, dyslipidaemia, predominantly hypertriglyceridaemia, might be a risk factor.

Adolescent↗
PubMed2002

Effect of hirudin vs heparin on haemostatic activity in patients with acute coronary syndromes; the GUSTO-IIb haemostasis substudy.

AIMS: We compared the effects of hirudin and heparin on thrombin generation and activity among 350 patients with acute coronary syndromes enrolled in the GUSTO-IIb trial. METHODS AND RESULTS: We obtained blood at baseline; at 4, 8, and 24h into infusion; at drug termination; and 6 and 24h after termination. We assayed for thrombin activity (fibrinopeptide A, activated protein C, thrombin-antithrombin complex), thrombin generation (prothrombin fragment 1.2), and platelet activation (platelet factor 4). Median baseline fibrinopeptide A and platelet factor 4 levels were elevated. Thrombin formation tended to increase with hirudin and decrease with heparin; by 8h into infusion, thrombin formation was significantly less for heparin (P<0.01). Most patients showed reduced thrombin activity and platelet activation during infusion of either agent. Hirudin-assigned patients had significantly lower fibrinopeptide A levels during infusion. Six h post-termination, both groups had increased thrombin activity. Thrombin formation was increased in heparin patients (P<0.0001), significantly more than with hirudin (P=0.005). Higher values of haemostasis markers tended to be associated with poorer 30-day outcomes. CONCLUSION: Although hirudin did not prevent generation of new thrombin, it appeared to inhibit thrombin activity more than did heparin and produced slower increases in thrombin formation after discontinuation. The reelevation of thrombotic markers after stopping intravenous antithrombin therapy and the tendency toward increased thrombotic events with post-treatment increases in marker levels suggest an ongoing, clinically significant prothrombotic state. These results raise the possibility of improving on current antithrombotics by preventing thrombin generation and thrombin activity and by sustained suppression of the prothrombotic state.

Acute Disease↗
PubMed2001

Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischemic stroke.

BACKGROUND: Activated protein C (APC) contributes to systemic anticoagulant and anti-inflammatory activities. APC may reduce organ damage by inhibiting thrombin generation and leukocyte activation. Neutrophils and cerebrovascular thrombosis contribute to ischemic neuronal injury, suggesting that APC may be a potential protective agent for stroke. METHODS AND RESULTS: We examined the effects of APC in a murine model of focal ischemia. After middle cerebral artery occlusion/reperfusion, the average survival time in controls was 13.6 hours. Animals that received purified human plasma-derived APC 2 mg/kg IV either 15 minutes before or 10 minutes after stroke induction survived 24 hours and were killed for neuropathological analysis. APC 2 mg/kg given before or after onset of ischemia restored cerebral blood flow, reduced brain infarct volume (59% to 69%; P:<0.003) and brain edema (50% to 61%; P:<0.05), eliminated brain infiltration with neutrophils, and reduced the number of fibrin-positive cerebral vessels by 57% (P:<0.05) and 25% (nonsignificant), respectively. The neuroprotective effect of APC was dose-dependent and associated with significant inhibition of ICAM-1 expression on ischemic cerebral blood vessels (eg, 61% inhibition with 2 mg/kg APC). Intracerebral bleeding was not observed with APC. CONCLUSIONS: APC exerts anti-inflammatory, antithrombotic, and neuroprotective effects in stroke. Central effects of APC are likely to be related to improved maintenance of the blood-brain barrier to neutrophils and to reduced microvascular obstructions and fibrin deposition.

Animals↗
PubMed2001

Plasma glucosylceramide deficiency as potential risk factor for venous thrombosis and modulator of anticoagulant protein C pathway.

To assess the relationship between venous thrombosis and plasma glucosylceramide (GlcCer) or phosphatidylethanolamine (PE), plasma levels of GlcCer and PE were determined for 70 venous thrombosis patients referred for evaluation and 70 healthy blood donors. The mean GlcCer level, but not the PE level, was lower in patients versus controls (4.9 vs 6.5 microg/mL [P =.0007] and 66 vs 71 microg/mL [P =.48], respectively). As a measure of relative risk, the odds ratio for deep vein thrombosis in subjects with GlcCer levels below the 10th percentile of controls was 5.7 (95% CI, 2.3-14). To assess the influence of glycolipids on anticoagulant response to activated protein C (APC):protein S in modified prothrombin time assays, the effects of depleting endogenous plasma GlcCer by glucocerebrosidase treatment or of adding exogenous purified GlcCer or other neutral glycolipids to plasma were tested. Glucocerebrosidase treatment reduced plasma sensitivity to APC:protein S in parallel with GlcCer reduction. Exogenously added GlcCer and the homologous Glc-containing globotriaosylceramide (Gb3Cer), but not galactosylceramide, dose-dependently prolonged clotting times of normal plasma in the presence, but not absence, of APC:protein S, which suggests that GlcCer or Gb3Cer can enhance protein C pathway anticoagulant activity. In studies using purified proteins, inactivation of factor Va by APC:protein S was enhanced by GlcCer alone and by GlcCer in multicomponent vesicles containing phosphatidylserine and phosphatidylcholine. These results suggest that the neutral glycolipids GlcCer and Gb3Cer may directly contribute to the anticoagulant activity of the protein C pathway and that deficiency of plasma GlcCer may be a risk factor for venous thrombosis. (Blood. 2001;97:1907-1914)

Activated Protein C Resistance↗
PubMed2001

Activated protein C and inflammation in human myocardium after heart surgery.

To assess possible interactions between inflammation and activation of the anticoagulant protein C system during post-ischemic reperfusion protein C, APC, neutrophil L-selectin expression and myocardial myeloperoxidase activity (MPO) were measured in 19 patients undergoing cardiopulmonary bypass. After reperfusion for 10 min, APC to protein C ratio (APC/PC) increased from pre-reperfusion value 1.43 +/- 0.12 (mean +/- SEM) to 2.25 +/- 0.29, p = 0.015. Negative correlations were observed between APC/PC and MPO activity (Spearman r -0.64, p = 0.007) and APC/PC and neutrophil L-selectin expression (r = -0.7, p = 0.007, demonstrating that post-ishemic protein C activation was associated with decreased neutrophil tissue sequestration. Thus, physiological protein C activation may be involved in regulation of the inflammatory injury during reperfusion of human ischemic coronary circulation.

Adult↗
PubMed2001

Potent anti-Trypanosoma cruzi activities of oxidosqualene cyclase inhibitors.

Trypanosoma cruzi is the protozoan agent that causes Chagas' disease, a major health problem in Latin America. Better drugs are needed to treat infected individuals. The sterol biosynthesis pathway is a potentially excellent target for drug therapy against T. cruzi. In this study, we investigated the antitrypanosomal activities of a series of compounds designed to inhibit a key enzyme in sterol biosynthesis, oxidosqualene cyclase. This enzyme converts 2,3-oxidosqualene to the tetracyclic product, lanosterol. The lead compound, N-(4E,8E)-5,9, 13-trimethyl-4,8, 12-tetradecatrien-1-ylpyridinium, is an electron-poor aromatic mimic of a monocyclized transition state or high-energy intermediate formed from oxidosqualene. This compound and 27 related compounds were tested against mammalian-stage T. cruzi, and 12 inhibited growth by 50% at concentrations below 25 nM. The lead compound was shown to cause an accumulation of oxidosqualene and decreased production of lanosterol and ergosterol, consistent with specific inhibition of the oxidosqualene cyclase. The data demonstrate potent anti-T. cruzi activity associated with inhibition of oxidosqualene cyclase.

3T3 Cells↗
PubMed2001

Anticoagulant dysfunction of human Arg352Trp-activated protein C caused by defective factor Va inactivation.

The dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin-thrombomodulin or alpha-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to approximately 65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.

Amino Acid Substitution↗
PubMed2001

Plasma lipoproteins, hemostasis and thrombosis.

Regulation of hemostasis and thrombosis involves numerous plasma factors that contribute to procoagulant and anticoagulant pathways. Lipid-containing surfaces provide sites where both procoagulant and anticoagulant enzymes, cofactors and substrates are assembled to express their activities. Plasma and lipoproteins can contribute to either procoagulant or anticoagulant reactions. Procoagulant lipids/lipoproteins include triglyceride-rich particles in plasma and oxidized low density lipoprotein (LDL) which can accelerate activation of prothrombin, factor X and factor VII. Potentially anticoagulant lipids and lipoproteins, each of which enhances inactivation of factor Va by activated protein C, include phosphatidylethanolamine, cardiolipin, the neutral glycosphingolipids glucosylceramide and Gb3 ceramide (CD77), and high density lipoprotein (HDL). Remarkably, treatment of hyperlipidemia with statins not only lowers lipids but also provides antithrombotic effects whose mechanisms remain to be clarified. We hypothesize that procoagulant and anticoagulant lipids and lipoproteins in plasma may contribute to a Yin-Yang balance that helps influence the up-regulation and down-regulation of thrombin generation.

Animals↗
PubMed2000

Chemical synthesis and spontaneous folding of a multidomain protein: anticoagulant microprotein S.

Because of recent high-yield native ligation techniques, chemical synthesis of larger multidomain bioactive proteins is rapidly coming within reach. Here we describe the total chemical synthesis of a designed "microprotein S," comprising the gamma-carboxyglutamic acid-rich module, the thrombin-sensitive module, and the first epidermal growth factor-like module of human plasma protein S (residues 1-116). Synthetic microprotein S expressed anticoagulant cofactor activity for activated protein C in the down-regulation of blood coagulation, and the anticoagulant activity of microprotein S was not neutralized by C4b-binding protein, a natural inhibitor of native protein S in plasma. The correct folding of this complex multidomain protein was enhanced compared with individual modules because the gamma-carboxyglutamic acid-rich module and the thrombin-sensitive module markedly facilitated correct folding of the first epidermal growth factor-like module compared with folding of the first epidermal growth factor-like module alone. These results demonstrate that total chemical synthesis of proteins offers an effective way to generate multidomain biologically active proteins.

1-Carboxyglutamic Acid↗
PubMed2000

Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S.

The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76-116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1 microM+ EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94 A (9.4 nm) to 85 A above the phospholipid surface (assuming kappa(2)=2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phospholipid-bound rhodamine was obtained upon association of protein S or protein S-C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S-C4b-binding protein with APC on the phospholipid surface. To confirm a direct interaction of EGF1 with APC, rhodamine was covalently attached to the alpha-N-terminus of EGF1, and binding of the labelled EGF1 to APC was directly demonstrated using FRET. The data suggested a separation between the active site of APC and the N-terminus of EGF1 of 76 A (kappa(2)=2/3), placing the APC-bound protein S-EGF1 close to, but above, the phospholipid surface and near the two EGF domains of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.

Amino Acid Sequence↗
PubMed2000

The autolysis loop of activated protein C interacts with factor Va and differentiates between the Arg506 and Arg306 cleavage sites.

The anticoagulant human plasma serine protease, activated protein C (APC), inactivates blood coagulation factors Va (FVa) and VIIIa. The so-called autolysis loop of APC (residues 301-316, equivalent to chymotrypsin [CHT] residues 142-153) has been hypothesized to bind FVa. In this study, site-directed mutagenesis was used to probe the role of the charged residues in this loop in interactions between APC and FVa. Residues Arg306 (147 CHT), Glu307, Lys308, Glu309, Lys311, Arg312, and Arg314 were each individually, or in selected combinations, mutated to Ala. The purified recombinant protein C mutants were characterized using activated partial thromboplastin time (APTT) clotting assays and FVa inactivation assays. Mutants 306A, 308A, 311A, 312A, and 314A had mildly reduced anticoagulant activity. Based on FVa inactivation assays and APTT assays using purified Gln506-FVa and plasma containing Gln506-FV, it appeared that these mutants were primarily impaired for cleavage of FVa at Arg506. Studies of the quadruple APC mutant (306A, 311A, 312A, and 314A) suggested that the autolysis loop provides for up to 15-fold discrimination of the Arg506 cleavage site relative to the Arg306 cleavage site. This study shows that the loop on APC of residues 306 to 314 defines an FVa binding site and accounts for much of the difference in cleavage rates at the 2 major cleavage sites in FVa. (Blood. 2000;96:585-593)

Arginine↗