Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock.

This study was designed to test the hypothesis that tissue factor pathway inhibitor (TFPI) plays a significant role in vivo in regulating coagulation that results from exposure of blood to tissue factor after vascular injury as in the case of gram negative sepsis. Highly purified recombinant TFPI (6 mg/kg) was administered either 30 min or 4 h after the start of a lethal intravenous Escherichia coli infusion in baboons. Early posttreatment of TFPI resulted in (a) permanent seven-day survivors (5/5) with significant improvement in quality of life, while the mean survival time for the controls (5/5) was 39.9 h (no survivors); and (b) significant attenuations of the coagulation response and various measures of cell injury, with significant reductions in pathology observed in E. coli sepsis target organs, including kidneys, adrenals, and lungs. TFPI administration did not affect the reduction in mean systemic arterial pressure, the increases in respiration and heart rate, or temperature changes associated with the bacterial infusion. TFPI treated E. coli infected baboons had significantly lower IL-6 levels than their phosphate buffered saline-treated controls, however tumor necrosis factor levels were similarly elevated in both groups. In contrast to the earlier 30-min treatment, the administration of TFPI at 4 h, i.e., 240 min, after the start of bacterial infusion resulted in prolongation of survival time, with 40% survival rate (2/5) and some attenuation of the coagulopathic response, especially in animals in which fibrinogen levels were above 10% of normal at the time of TFPI administration. Results provide evidence for the significance of tissue factor and tissue factor pathway inhibitor in bacterial sepsis, and suggest a role for blood coagulation in the regulation of the inflammatory response.

Fibrinogen mediates platelet-polymorphonuclear leukocyte cooperation during immune-complex glomerulonephritis in rats.

The metabolic and functional alterations which occur during the acute phase of nephrotoxic nephritis (NTN) in rats, a model of immune-mediated glomerulonephritis, result from a cooperative interaction between PMNs and platelets (PLTs). In consequence, we hypothesized that fibrinogen (Fg) might play a critical role in this process and, accordingly, we found that defibrination of animals decreased both the acute phase proteinuria in NTN (approximately 70%) as well as the influx of PLTs and PMNs into the glomerulus (approximately 40-50%). In contrast, blockade of the PLT Fg receptor, alpha IIb beta 3, with the RGD peptidomimetic SC-49992 decreased proteinuria (approximately 90%) without substantially altering the influx of PMNs or PLTs. Immunocytochemistry showed a marked increase in beta 3 integrin expression in inflamed glomeruli which was prevented either by PMN or PLT depletion before disease induction. FACS and immunocytochemical analysis of glomerular cell dissociates demonstrated that beta 3 integrin expression was predominantly on intraglomerular PLTs. In vitro, activated PLTs stimulated the PMN respiratory burst, an interaction which could be inhibited by Fg receptor blockade. In sum, acute NTN is accompanied by a marked increase in glomerular beta 3 integrin expression predominantly due to the influx of PLTs which localize to the glomerulus in a PMN-dependent fashion. Fg appears to serve a major role as a coactivating stimulus for PLT-PMNs in situ via alpha IIb beta 3, potentially mediating the PMN respiratory burst which contributes to proteinuria. Fg may also play a subsidiary role in PMN/PLT comigration.

Platelet GPIIb/IIIa receptor inhibition by SC-49992 prevents thrombosis and rethrombosis in the canine carotid artery.

OBJECTIVE: Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptors represent the final common pathway for aggregation. GPIIb/IIIa inhibition with antibodies or RGD peptides prevents arterial thrombosis. The present study examined the ability of SC-49992 (SC), a GPIIb/IIIa receptor antagonist, to prevent thrombosis in a canine model of carotid artery thrombosis. METHODS: Both carotid arteries of anaesthetised dogs were instrumented with Doppler probes. A 300 microA current was applied to the intimal surface of the right carotid artery via an intraluminal electrode; time to occlusive thrombus formation was noted. SC (30 and 60 micrograms/.kg-1 x min-1, intravenously) or saline was infused for 240 min. The procedure for thrombus formation was repeated after 60 min of drug infusion for the left carotid artery. RESULTS: SC did not alter heart rate or blood pressure. Frequency of occlusive thrombus formation was reduced or prevented in a dose dependent manner (control = 100%, n = 12; SC 30 micrograms = 50%, n = 6; SC 60 micrograms = 0%, n = 6; p < 0.05). SC resulted in a reduction in thrombus weight (p < 0.05) v control. Ex vivo platelet aggregation to ADP and arachidonic acid was inhibited. Platelet reactivity remained inhibited 60 min after cessation of SC infusion. In a second group of animals, a carotid artery thrombus was formed and lysed with administration of anisoylated plasminogen streptokinase activator complex (0.05 U.kg-1). SC (60 micrograms.kg-1 x min-1, intravenously, n = 6) or saline (n = 6) was infused for 240 min. In dogs receiving saline there was an 83% rate of rethrombosis; none of the SC treated animals had reocclusion after recanalisation (p < 0.05). CONCLUSIONS: SC-49992 inhibits ex vivo platelet aggregation, prevents occlusive thrombus formation in a canine model of arterial thrombosis, and prevents rethrombosis after thrombolysis. The data are consistent with results obtained with murine monoclonal antibodies directed against the platelet GPIIb/IIIa receptor.

A mimetic of the RGDF-peptide [arginine-glycine-aspartic acid-phenylalanine] blocks aggregation and flow-induced platelet deposition on severely injured stenotic arterial wall. Effects on different animal models and in humans.

8-Guanidino-octanoyl-aspartic acid-phenylalanine (SC-49992), a mimetic of the tetrapeptide arginine-glycine-aspartic acid-phelylalanine, inhibits fibrinogen and vitronectin binding to GP IIb/IIIa. SC-49992 effects on impedance and optical aggregation were compared in different species (human, porcine and dog), SC-49992 induced significant inhibition both in whole blood and PRP aggregation, in all species; however, porcine platelets had a SC-49992 IC50 = 2.5 mM while human and dog platelets had a significant lower IC50 (1 microM and 1.5 microM, respectively). Inhibition of flow-mediated platelet deposition on severely injured vessels was studied in porcine blood at high and low shear rates for 5 minutes. Additionally, studies were performed in vessels with 80% stenosis. SC-49992 reduced platelet deposition both at high and low shear rate in parallel streamlined flow and in stenotic conditions. The lower affinity of porcine platelets for SC-49992 seems to be due to a species difference at the GPIIb/IIIa receptor RGD-sequence level.

A new method for measurement of plasma concentration of orally active glycoprotein IIb/IIIa antagonists.

A bioassay for determining concentrations of antiplatelet compounds in plasma or aqueous solution has been developed. The method uses an aliquot of plasma from treated animals to inhibit collagen-induced platelet aggregation in pooled platelet-rich plasma (PRP) obtained from donor dogs. The concentration in plasma from treated animals was estimated using a standard curve of inhibition established using plasma from untreated animals which had been spiked with known amounts of compound. For independent validation, plasma concentrations of certain compounds were determined in identical dog plasma samples by both bioassay and HPLC. Results from the two methods were concordant. The bioassay provides an accurate and sensitive method for measuring antiplatelet activity without the need for extraction of plasma samples and may be used to measure activity in any solution which is compatible with PRP. This assay is routinely used to provide an estimate of absorption of prodrugs and systemic conversion to active compound after oral dosing. Some of the compounds of interest are ester-acid pairs with the inactive ester prodrug being cleaved to the active acid following administration. Compounds were administered orally (ester) or IV (acid) and blood samples were taken periodically for 24 hours. Plasma concentration of active moiety was determined for each time point and the area under the curve (AUC) of concentration vs. time was calculated. Comparing the AUCs for oral and IV routes of administration yielded the Oral Systemic Activity (OSA), a measure of active compound available after oral dosing.

SC-49992--a potent and specific inhibitor of platelet aggregation.

Fibrinogen binding is required for platelet aggregation and subsequent thrombus formation. SC-49992 (SC), an RGDF mimetic, is a potent and specific inhibitor of the binding of fibrinogen to its receptor on activated platelets, glycoprotein IIb/IIIa (IC50 0.7 microM). SC was more potent (1-5 microM) than either RGDS, RGDF or the gamma chain dodecapeptide in blocking platelet aggregation to a variety of agonists in both dog and human platelet rich plasma. SC was more potent as an inhibitor of GP IIb/IIIa on platelets than it was against other integrin and non-integrin receptors, including the RGD-dependent vitronectin receptor and other non-RGD-dependent integrins such as CDII/CD18. SC had little effect on ristocetin induced agglutination. SC blocked ex vivo collagen induced aggregation in dogs and collagen induced thrombocytopenia in rats. These data suggest that elimination of the Arg-NH2 and the Arg-Gly amide bond of RGDF provided increased inhibitory potency and specificity. This structural modification may be of value in the development of other more potent RGDF mimetics for the inhibition of platelet aggregation.

Species variation in the effect of glycoprotein IIb/IIIa antagonists on inhibition of platelet aggregation.

Differences exist between platelets of different species in their reaction to pharmaceutical agents, such as inhibitors of platelet aggregation. Understanding these differences is critical in the interpretation of data from experimental animal models of thrombosis. Platelet aggregation, essential in the hemostatic process, requires that fibrinogen (fgn) bind to activated platelets. Analogs of Arginine-Glycine-Aspartic acid-Phenylalanine (RGDF), a peptide sequence of fgn, block fgn binding to its receptor known as glycoprotein (GP) IIb/IIIa on activated platelets and prevent aggregation. We studied the inhibition resulting from Arginine-Glycine-Aspartic acid-Serine (RGDS) and two analogs of RGDF, (SC-46749 and SC-47643) on aggregation of human, rat, guinea pig, dog, and rhesus monkey platelets in vitro using ADP as the agonist. The inhibitory potency of RGDS, SC-46749, ad SC-47643 was species dependent. The rank order of potency was rhesus monkey, dogs, and human followed by guinea pig and rat. In order to study the relative inactivity of the compounds in rat platelets compared to human, we diluted rat platelet-rich plasma (PRP) to yield platelet levels approximating that of humans. Platelet inhibition was not significantly changed in diluted rat PRP nor did changing concentration appear to affect activity in human PRP. Our data suggest that the platelet response of some species may better represent human response with regard to inhibition of GP IIb/IIIa by (RGDX) analogs.

Influence of renal lipoxygenase activity on the renal vascular response to arachidonic acid.

Studies were undertaken to clarify the effects of close arterial injections of arachidonic acid (AA) on renal blood flow in anesthetized dogs. In some dogs, injection of 4 mg of AA into the renal artery produces only renal vasodilation, whereas in other dogs similar injections lead to biphasic responses in which vasodilation is preceded by transient vasoconstriction. In the present experiments the cyclooxygenase inhibitor ibuprofen blocked vasodilator responses, suggesting that these were mediated by conversion of the precursor to prostaglandins. However, ibuprofen did not block the constrictor phase of the response in those animals that exhibited biphasic responses, suggesting that this phase was not mediated by prostaglandins. Administration of agents that inhibit lipoxygenase and cytochrome P-450 enzymes blocked the constrictor phase, suggesting that this portion of the responses was associated with conversion of the precursor to hydroxylated eicosanoids. An additional observation from these studies was that the frequency of occurrence of biphasic responses to intrarenal AA injections in water-deprived dogs was significantly greater than that found in non-water-deprived dogs, suggesting a connection between hydration state and the activity of nonprostaglandin pathways for AA metabolism in the canine kidney.

Actions of prostaglandins in peripheral vascular beds.

The prostaglandins comprise a large family of substances that includes primary prostaglandins (PGE2, F2 alpha, D2) prostacyclin (PGI2), and thromboxanes (TxA2, B2), all of which exhibit some vascular activity. The activity of each prostaglandin may be species- and organ-dependent, and the type of prostaglandin produced in a tissue is often dependent on the presence of terminal enzyme systems in that tissue. This paper describes the effects of certain members of the prostaglandin family on the mesenteric and renal vascular beds of anesthetized dogs. In the kidney, arachidonic acid, PGE2, D2, and I2 all produce vasodilation when studied by bolus injection techniques. PGF2 alpha is relatively inactive. In the mesenteric vascular bed arachidonate, PGE2 and PGI2 are vasodilators whereas PGD2 and PGF2 are vasoconstrictors. The direct actions of thromboxanes A2 and the endoperoxide PGH2 are uncertain because of their very short half-lives in plasma. Infusion of prostaglandins E2 and I2 into the left ventricle produces vasodilation in both the kidney and the gut. In the mesenteric vascular bed, however, vasodilation during PGE2 (but not PGI2) infusion is a transient phenomenon and the blood flow returns to control levels within the first few minutes of infusion. The vascular actions of prostaglandins are therefore complex and are dependent not only on organ and species, but on experimental technique as well.