Inhibition Of Collagen-induced Aggregation Research Articles

Left Ventricular Assist Device Induced Coagulation and Platelet Activation and the Effect of Current Anticoagulant Therapy Regimen

Abstract 48530 Background:Left ventricular assist devices (LVAD) are used to improve circulation in heart failure patients until an available transplant. Despite the benefits of these pumps, they have also been shown to cause a higher rate of adverse effects than the customary medical treatment. Specifically, these implants have been shown to heighten the incidence of both bleeding events (such as intestinal bleeds) and thromboembolic events. Previous studies have attributed these episodes to shear stress caused by blood flow through the pump and to contact of the blood with the artificial surfaces of the device. Contact of blood with artificial surfaces can activate the clotting cascade, while shear stress can directly activate platelets and can impact the functionality of von Willebrand factor. Therefore, a testing regimen that can predict the occurrence of adverse events could lead to enhanced clinical benefit of ventricular assist devices. Methods:Patients implanted with the HeartMate II LVAD at Loyola University Medical Center were enrolled in the study. Blood samples are drawn on a monthly basis and at time of an adverse event. These blood samples were analyzed using flow cytometry to assess P-selectin expression and PAC-1 binding (activated GPIIb/IIIa), the ICHOR PlateletWorks assay (Helena Laboratories; Beaumont, TX) and thrombelastography using the Platelet Mapping assay (Haemoscope; Niles, IL). Patient demographic and clinical data was obtained from medical records. Additionally, blood drawn from 5 individuals per group of warfarin alone, aspirin alone, and normal individuals served as controls. Results:Patients included 15 males and 5 females with a mean age of 53.3 ±13.5 years (range: 23.5 to 70 years) at the time of enrollment. Of the patients 35% were diabetic, 36.7 % were hypertensive, and 45% were hyperlipidemic. Of the 20 patients 18 were on warfarin (mean starting dose of 4.6±2.5 mg) and 19 of 20 patients were on aspirin (81–325 mg/day). Additionally, 60% of patients were treated with an ACE inhibitor, 80% with a β-blocker and 80% with a diuretic. Of the 20 patients, 2 patients had experienced adverse events during the study period. One had a small bowel bleed requiring multiple transfusions. The second had a 3-day GI bleed requiring multi-unit transfusion. By flow cytometry, both patients with bleeding episodes exhibited less of an activation response to platelet agonists ADP, arachidonic acid and TRAP in terms of P-selectin expression and PAC-1 binding than the mean of the stable LVAD group or the normal individuals. In the Platelet Mapping assay, the stable LVAD population exhibited a 31.5 ± 18.4% (mean ± SD) inhibition of ADP-induced activation (p<0.05 vs normals at 15.1 ± 4.8%); the patient with the small bowel bleed exhibited a 65.6% inhibition. In the ICHOR PlateletWorks assay, the stable LVAD population exhibited a 37.3 ± 19.7% inhibition of collagen-induced aggregation (p=0.004 vs normals at 7.9 ± 1.9%); the patients experiencing bleeding events had inhibition levels of 63.7% and 74.2%, respectively. In the PlateletWorks assay, while the stable LVAD population exhibited a 21.2 ± 18.2% inhibition of ADP-induced aggregation (p<0.004 vs normals at 6.1 ± 2.6%), the patient with the GI bleed exhibited an inhibition level of 72.6%. In the PlateletWorks and PlateletMapping assays, several patients exhibited results indicative of low platelet reactivity, but had not experienced a hemorrhagic event to date. These patients will be followed with periodic monitoring of platelet function to determine the predictive nature of each assay for any coagulation disorder. Discussion:Through these studies a baseline of normal platelet function has been established for our LVAD patient population. Further, while early in our evaluation the selected assays of PlateletWorks, PlateletMapping, and flow cytometry suggest that it may be possible to identify patients at risk of LVAD-associated bleeding complications. Disclosures:No relevant conflicts of interest to declare.

Left Ventricular Assist Device Induced Coagulation and Platelet Activation and the Effect of Current Anticoagulant Therapy Regimen

Abstract 5201 Background:Left ventricular assist devices (LVAD) are used to improve circulation in heart failure patients until an available transplant. Despite the benefits of these pumps, they have also been shown to cause a higher rate of adverse effects than the customary medical treatment. Specifically, these implants have been shown to heighten the incidence of both bleeding events (such as intestinal bleeds) and thromboembolic events. Previous studies have attributed these episodes to shear stress caused by blood flow through the pump and to contact of the blood with the artificial surfaces of the device. Contact of blood with artificial surfaces can activate the clotting cascade, while shear stress can directly activate platelets and can impact the functionality of von Willebrand factor. Therefore, a testing regimen that can predict the occurrence of adverse events could lead to enhanced clinical benefit of ventricular assist devices. Methods:Patients implanted with the HeartMate II LVAD at Loyola University Medical Center were enrolled in the study. Blood samples are drawn on a monthly basis and at time of an adverse event. These blood samples were analyzed using flow cytometry to assess P-selectin expression and PAC-1 binding (activated GPIIb/IIIa), the ICHOR PlateletWorks assay (Helena Laboratories; Beaumont, TX) and thrombelastography using the Platelet Mapping assay (Haemoscope; Niles, IL). Patient demographic and clinical data was obtained from medical records. Additionally, blood drawn from 5 individuals per group of warfarin alone, aspirin alone, and normal individuals served as controls. Results:Patients included 15 males and 5 females with a mean age of 53.3 ±13.5 years (range: 23.5 to 70 years) at the time of enrollment. Of the patients 35% were diabetic, 36.7 % were hypertensive, and 45% were hyperlipidemic. Of the 20 patients 18 were on warfarin (mean starting dose of 4.6±2.5 mg) and 19 of 20 patients were on aspirin (81–325 mg/day). Additionally, 60% of patients were treated with an ACE inhibitor, 80% with a β-blocker and 80% with a diuretic. Of the 20 patients, 2 patients had experienced adverse events during the study period. One had a small bowel bleed requiring multiple transfusions. The second had a 3-day GI bleed requiring multi-unit transfusion. By flow cytometry, both patients with bleeding episodes exhibited less of an activation response to platelet agonists ADP, arachidonic acid and TRAP in terms of P-selectin expression and PAC-1 binding than the mean of the stable LVAD group or the normal individuals. In the Platelet Mapping assay, the stable LVAD population exhibited a 31.5 ± 18.4% (mean ± SD) inhibition of ADP-induced activation (p<0.05 vs normals at 15.1 ± 4.8%); the patient with the small bowel bleed exhibited a 65.6% inhibition. In the ICHOR PlateletWorks assay, the stable LVAD population exhibited a 37.3 ± 19.7% inhibition of collagen-induced aggregation (p=0.004 vs normals at 7.9 ± 1.9%); the patients experiencing bleeding events had inhibition levels of 63.7% and 74.2%, respectively. In the PlateletWorks assay, while the stable LVAD population exhibited a 21.2 ± 18.2% inhibition of ADP-induced aggregation (p<0.004 vs normals at 6.1 ± 2.6%), the patient with the GI bleed exhibited an inhibition level of 72.6%. In the PlateletWorks and PlateletMapping assays, several patients exhibited results indicative of low platelet reactivity, but had not experienced a hemorrhagic event to date. These patients will be followed with periodic monitoring of platelet function to determine the predictive nature of each assay for any coagulation disorder. Discussion:Through these studies a baseline of normal platelet function has been established for our LVAD patient population. Further, while early in our evaluation the selected assays of PlateletWorks, PlateletMapping, and flow cytometry suggest that it may be possible to identify patients at risk of LVAD-associated bleeding complications. Disclosures:No relevant conflicts of interest to declare.

Carbon monoxide released by CORM-3 inhibits human platelets by a mechanism independent of soluble guanylate cyclase

Carbon monoxide (CO) modulates several physiological functions through activation of a cGMP-dependent pathway similar to that of nitric oxide (NO). Here we investigated the possible involvement of soluble guanylate cyclase in the anti-aggregatory effect of micromolar concentrations of CO released by a novel, water-soluble, CO releasing molecule (CORM) in human platelets. Human platelet aggregation was induced by collagen or thrombin, and the effects of CO releasing molecule (CORM-3) and an NO donor on platelet aggregation were compared. CORM-3 liberated CO in a time- and concentration-dependent manner as evidenced by the formation of carbon monoxy myoglobin (MbCO) using a spectrophotometric assay. When added to washed platelets, CORM-3 (10-300 microM) inhibited collagen- and thrombin-induced aggregation in a concentration-dependent manner. The anti-aggregatory effect of CORM-3 was reversed by deoxy-Mb (50 microM). Interestingly, in the presence of an inhibitor of guanylate cyclase (ODQ, 5 microM), inhibition of collagen-induced aggregation by CORM-3 was not blocked but potentiated. Under the same experimental conditions, inhibition of platelet aggregation by an NO donor (SNAP, 1 microM) was prevented by ODQ. In collagen-induced or thrombin-induced platelet aggregation, a stimulator of guanylate cyclase (YC-1, 0.3 microM) did not alter the effect of CORM-3, whereas it markedly potentiated the inhibition of platelet aggregation mediated by SNAP. Notably, CORM-3-induced inhibition of platelet aggregation was of similar degree when platelets were activated by a low (20 mU/ml) or by high concentration of thrombin (100-200 mU/ml), whereas NO donors (SNP and SNAP)- or carbaprostacylin (cPGI(2))-induced effects were considerably attenuated when platelets were activated by high concentrations of thrombin. Inhibition of platelet aggregation by CO released by a novel, water-soluble CORM is not mediated by activation of soluble guanylate cyclase. In contrast to NO and PGI(2), CO effectively inhibits platelets even when cells are activated excessively. We suggest that despite the fact that CO is not a potent inhibitor of platelet activation, it may gain importance when NO and PGI(2) alone are insufficient to overcome excessive platelet activation.

Comparison of Rapid Platelet Function Assay with Whole Blood Platelet Impedance Aggregometry for the Definition of Aspirin Resistance.

The reported incidence of aspirin (ASA) resistance ranges from 5 to 30%. Various platelet function assays have been employed to detect aspirin resistance in patients with cardio- and cerebrovascular disease. Such a high proposed incidence of ASA resistance poses a critical need for a rapid point-of -care (POC) platelet function test. Unfortunately, no uniformly accepted definition of ASA resistance exists. Platelet aggregation studies that have been used to define ASA resistance are time consuming and require special technical expertise. The Ultegra Rapid Platelet Function -ASA (RPFA-ASA) has been developed as a POC test that is performed without sample processing. This optical method measures agglutination of fibrinogen-coated beads upon platelet activation with arachidonic acid. In the presence of aspirin effect, however, the agglutination of the beads is inhibited. The described cutoff of ≥ 550 Aspirin Reaction Units (ARU) is termed non-responsiveness to ASA based on a preclinical study and subsequent correlation with epinephrine-induced platelet aggregation in platelet rich plasma. Since RPFA-ASA uses whole blood, we validated its performance characteristics against a classic whole blood platelet aggregation assay (WBA). We studied 50 healthy volunteers, aged 25–75 (24 men, 26 women) with normal CBC, who had not ingested anti-platelet drugs for 14 days prior to the study. Baseline studies included WBA (dual channel aggregometer, Chrono-log Inc., Havertown, PA) using both arachidonic acid (AA -0.5; 0.25 mM) and collagen (1; 2 μg/mL) as well as an RPFA-ASA assay (Accumetrics Inc., San Diego, CA). These studies were repeated after 3 days of ASA (325 mg/d) intake. Based on a review of the literature, we defined an adequate ASA response as a completely inhibited AA-induced platelet aggregation and at least 30% inhibition of collagen-induced aggregation (both concentrations of the agonist). Thus, those with < 30% inhibition of aggregation response to collagen were considered ASA resistant. Eleven subjects were ASA resistant by WBA (20%; 8 females and 3 males (aged 25–63). In contrast, since all 50 subjects achieved ARU values of < 550 ARU, none were recognized as an ASA non-responder by the RPFA-ASA. While the current cutoff of < 550 ARU posed by the Ultegra RPFA-ASA does identify those who have taken ASA, the assay is unable to recognize ASA non-responders. Thus, based on these data, the appropriate cutoff for the recognition of ASA resistance by the RPFA-ASA should be re-adjusted to a significantly lower level to ensure appropriate assay results.

Structure of rhodocetin reveals noncovalently bound heterodimer interface

Rhodocetin is a unique heterodimer consisting of alpha- and beta-subunits of 133 and 129 residues, respectively. The molecule, purified from the crude venom of the Malayan pit viper, Calloselasma rhodostoma, functions as an inhibitor of collagen-induced aggregation. Rhodocetin has been shown to have activity only when present as a dimer. The dimer is formed without an intersubunit disulfide bridge, unlike all the other Ca(2+)-dependent lectin-like proteins. We report here the 1.9 A resolution structure of rhodocetin, which reveals the compensatory interactions that occur in the absence of the disulfide bridge to preserve activity.

Effects of lipid-esterified conjugated linoleic acid isomers on platelet function: evidence for stimulation of platelet phospholipase activity

The effects of four conjugated linoleic acid (CLA) isomers on in vitro collagen-induced human platelet aggregation and thromboxane (TXB 2, the inactive metabolite of the proaggregatory TXA 2) production were examined. As the free fatty acid (FFA), 9t, 11t-CLA was the most effective inhibitor of these two processes (I 50s of 2.2 and 4 μM, respectively) and the 9c, 11c-CLA was the least effective (I 50s of 8.3 and 37 μM) of the isomers tested. When platelets were preesterified with either 25 μM 9t, 11t-CLA or 9c, 11c-CLA, CLA incorporation in total platelet lipids increased from 0.24% to 0.31% and 0.38%, and most of this increase was found to be in the phosphatidyl choline and phosphatidyl ethanolamine subclasses. The decrease in arachidonic acid (AA) content in total fatty acids or phospholipids was an order of magnitude greater. Furthermore, no significant differences between platelets prelabeled with either 9t, 11t- or 9c, 11c-CLA in the inhibition of collagen-induced aggregation and TXB 2 formation were observed. However, platelets prelabeled with 9c, 11c-CLA stimulated basal TXB 2 production (4-fold) which was not observed with platelets pretreated with either 9t, 11t-CLA, linoleic acid or stearic acid. This enhancement was associated with a 2.4–5-fold increase in the release of endogenous AA. Our results suggest that the presence of a conjugated cis, cis double bond appears to change the lipid environment sufficiently to stimulate the basal platelet phospholipase activity, which in turn increases the formation of TXB 2.

NITRIC OXIDE PRESERVES PLATELET FUNCTION DURING IN VITRO VENTRICULAR ASSIST DEVICE CIRCULATION

Introduction: Ventricular assist devices (VADs) have been used as an effective bridge to transplantation in patients with advanced heart failure. However, severe bleeding and thromboembolism limit their long-term use. Nitric Oxide (NO) has been evaluated as a potent agent that prevents platelet aggregation and preserves platelet function. Their use however, has not been characterized in VAD circulation. We hypothesize that s-nitrosoglutathione (GSNO), a NO donor, will preserve platelet function during in vitro VAD circulation. Methods: To determine an effective concentration of GSNO for inhibition of platelet activation, a dose-response measurement was first performed with GSNO to measure inhibition of whole blood platelet aggregation. Next, the concentration of GSNO that yielded approximately 50% platelet inhibition (225.8mg) was added to 450 ml of fresh human whole blood circulated through an in vitro nonpulsatile centrifugal VAD circuit for 72 hours. Physiological conditions were monitored and maintained. Total nitrogen oxides (NOT) were measured throughout the 72 hours of circulation using a NO analyzer. Results: The dose-response curve demonstrated that 1000M GSNO inhibited both ristocetin and collagen induced platelet aggregation by 52.0% and 56.8%, respectively. However, as the GSNO concentration was increased past 1000M, inhibition of collagen-induced aggregation was progressively greater than ristocetin-induced aggregation. After addition of GSNO to the in vitro VAD, ristocetin induced aggregation decreased initially by 57.3%. This inhibitory effect on aggregation was maintained during the first 3 hours of VAD circulation, and thereafter aggregation was preserved through 48 hours of VAD circulation. Collagen induced aggregation decreased initially by 46.8% after addition of GSNO. This inhibitory effect was maintained during the first 2 hours of VAD circulation, and thereafter aggregation was preserved through 24 hours of VAD circulation. In control VADs, NOT remained constant at 15.4uM whereas the NOT level in VADs treated with GSNO was 821.4. Conclusion: The ability of GSNO to differentially inhibit agonist specific platelet activation suggests that its mechanism of action is receptor-specific. Our findings that NOT was maintained at elevated levels throughout circulation suggest that GSNO is an effective NO donor. Nitric Oxide may be clinically useful in preserving platelet function during prolonged VAD use.

Effect of prostaglandin (PG) E 1 and its initial metabolites on neutrophil-induced inhibition of human platelet aggregation

We have investigated the effects of PGE 1, 15-keto-PGE 1, 15-keto-13,14-dihydro-PGE 1 and 13,14-dihydro-PGE 1 on inhibition of human platelet aggregation by rat peritoneal neutrophils (RPN) and human polymorphonuclear neutrophils (PMN). Both RPN and PMN are known to synthesize nitric oxide (NO). In the presence of a threshold concentration of RPN or PMN the inhibitory effects of PGE 1 and 13,14-dihydro-PGE 1 on thrombin- or collagen-induced platelet aggregation were significantly increased as compared to the absence of cells, while 15-keto-PGE 1 and 15-keto-13,14-dihydro-PGE 1 were inactive. Oxyhemoglobin (oxy-Hb) abolished the synergistic effect of RPN and either PGE 1 or 13,14-dihydro-PGE 1 on thrombin-induced platelet aggregation, but under the experimental conditions used had much less effect on inhibition of collagen-induced aggregation. Potentiation of the antiaggregatory effect of PGE 1 and 13,14-dihydro-PGE 1 by NO might contribute to the therapeutic efficacy of exogenous PGE 1. This view is supported by the fact that plasma levels of PGE 1 and its active metabolite in patients receiving infusions of PGE 1 for treatment of peripheral arterial occlusive disease are in the order of magnitude acting synergistically with neutrophil-derived NO, while direct inhibition of platelet aggregation requires considerably higher concentrations of PGE 1 and 13,14-dihydro-PGE 1.

Lupus anticoagulant antibodies inhibit collagen-induced adhesion and aggregation of human platelets in vitro.

The effect of circulating lupus anticoagulant on platelet interaction with collagen and other proteins was tested, with the aim of understanding the role of membrane phospholipids in platelet function. Plasma samples from 26 systemic lupus erythematosus (SLE) patients, containing circulating lupus anticoagulant (LAC), were examined for their effect on adhesion and aggregation of normal human platelets. We find that SLE plasma, but not normal plasma, inhibits platelet adhesion to collagen in a concentration-dependent manner. At a plasma concentration of 1% the inhibition was 73 +/- 9% (mean +/- SD). In sharp contrast, there was no effect on platelet adhesion to fibronectin. Purified IgG from the same plasma samples also had an inhibitory effect. At 15 micrograms/ml (comparable in IgG concentration to 0.1% plasma) it inhibited adhesion to collagen by 33 +/- 11%. Inhibition could be abolished by preincubation of the LAC-containing plasma with cardiolipin (CL), phosphatidylinositol (PI), and, to a lesser extent, phosphatidylserine (PS) but not with phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Inhibition could also be abolished by preincubation of the LAC-containing plasma with a 10-fold excess of washed normal platelets. The effect of 1% LAC plasma on platelet aggregation was as striking, showing 79 +/- 26% inhibition of collagen-induced aggregation, and it could also be abolished by preincubation of the LAC plasma with cardiolipin. In contrast, the effect of LAC plasma on thrombin-induced aggregation was rather modest. Our results indicate that antiphospholipid antibodies interfere with platelet adhesion and stimulation by collagen in vitro and point to an important role of external plasma membrane phospholipids, particularly PI, in collagen-induced platelet activation.

Antiplatelet effect of butylidenephthalide

Butylidenephthalide inhibited, inhibited, in a dose-dependent manner, the aggregation and release reaction of washed rabbit platelets induced by collagen and arachidonic acid. Butylidenephthalide also inhibited slightly the platelet aggregation induced by PAF and ADP, but not that by thrombin or ionophore A23187. Thromboxane B 2 formation caused by collagen, arachidonic acid, thrombin and ionophore A23187 was in each case markedly inhibited by butylidenephthalide. Butylidenephthalide inhibited the aggregation of ADP-refractory platelets, thrombin-degranulated platelets, chymotrypsin-treated platelets and platelets in the presence of creatine phosphate/creatine phosphokinase. Its inhibition of collagen-induced aggregation was more marked at lower Ca 2+ concentrations in the medium. The aggregability of platelets inhibited by butylidenephthalide could be recovered after the washing of platelets. In human platelet-rich plasma, butylidenephthalide and indomethacin prevented the secondary aggregation and blocked ATP release from platelets induced by epinephrine. Prostaglandin E 2 formed by the incubation of guinea-pig lung homogenate with arachiodonic acid could be inhibited by butylidenephthalide, indomethacin and aspirin. It is concluded that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be due partly to interference with calcium mobilization.

The In Vitro and In Vivo Effects of Isoflurane and Nitrous Oxide on Platelet Aggregation

In view of the possible antiaggregation effects of newer general anesthetics we investigated the in vitro and in vivo effects of isoflurane and nitrous oxide on platelet aggregation. Platelets obtained from 18 healthy volunteers, were exposed in vitro for 30 min in a closed chamber to oxygen-carbon dioxide (90%, 5%) (control), oxygen-carbon dioxide-nitrous oxide (80%), or oxygen-carbon dioxide-isoflurane (1.5%) with or without nitrous oxide (80%). Samples were tested in ADP- and collagen-induced aggregation tests. Both nitrous oxide and isoflurane produced statistically significant inhibition of ADP-induced aggregation. Inhibition of collagen-induced aggregation was not statistically significant. In 18 patients who received nitrous oxide (3 L/min) and isoflurane (1-2%) during anesthesia, platelet aggregation was reduced significantly. We conclude that both nitrous oxide and isoflurane cause moderate but statistically significant inhibition of platelet aggregation that may be clinically important in some patients.

The inhibitory effects of ferriprotoporphyrin IX on platelet aggregation and release of serotonin

Ferriprotoporphyrin IX (FP) dissolved in 0.025N NaoH in concentrations of 0.01 – 0.04uM/ul platelet suspension competitively inhibited platelet aggregation induced by a low concentration of collagen. 14C-serotonin release was also inhibited. Higher concentrations of collagen overcame the aggregation inhibition. A similar pattern of results was obtained with thrombin-, and arachidonic acid-induced aggregation and release. With ristocetin. there was little inhibition of aggregation although serotonin release was inhibited. ADP-induced aggregation was partially inhibited except at FP concentrations of 0.91 uM/ul. FP caused only platelet shape change and serotonin release of up to 8.1%. These changes were not associated with significant platelet lysis and could also not be attributed to pH or temperature changes. There was no inhibition of collagen-induced aggregation in PRP, but FP precipitates aggregated washed platelets and caused serotonin release. These results show that FP in solution inhibited platelet aggregation induced by the different agents studied. It did not interfere with platelet agglut ination induced by ristocetin. The mechanism(s) of aggregation inhibition remains to be clarified.

Inhibition of Platelet Aggregation by Human Placenta

Human placental cytosol inhibits platelet aggregation induced by high doses of collagen. The aim of this study was to investigate whether this anti-aggregating activity was caused only by the presence of various activities already described in the placenta (an ADP-consuming enzyme, a fatty acid cyclooxygenase inhibitor, and a thromboxane synthetase inhibitor) or whether another factor was present. Heating the cytosol at 50 degrees C for 6 min destroyed the inhibitor of collagen-induced aggregation. ADPase and the AA pathway inhibitors were not modified by this treatment. We therefore show the presence of an additional anti-aggregating factor: it is destroyed by heating at 50 degrees C. We also tested for the presence of an inhibitor of AA release in the placental cytosol using three different methods (rabbit platelets in PRP, washed rabbit platelets, and NRK fibroblasts) but no inhibition could be evidenced. We conclude that this new anti-aggregating factor, which is probably a protein, acts neither through AA release inhibition nor AA cascade inhibition.

Role of calmodulin in platelet aggregation. Structure-activity relationship of calmodulin antagonists.

Two series of derivatives of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), including a dechlorinated analog of W-7 (W-5) and various aminoalkyl chain analogs of W-7 (A-3, A-4, A-5, I-240, A-6) were synthesized and their structure-activity relationships with calmodulin antagonistic actions and their potencies in inhibiting human platelet aggregation in vitro were investigated. Their binding affinities to calmodulin in the presence of 100 microM Ca2+ were dependent both on the chlorination of the naphthalene ring and on the length of aminoalkyl chain. The ability of these derivatives to inhibit Ca2+-dependent phosphorylation of 20,000-dalton myosin light chain from platelets correlated well with the magnitude of their binding affinity to calmodulin. W-7(10-100 microM) inhibited in a dose-dependent manner platelet aggregation induced by collagen (2 micrograms/ml), ADP (5 microM), epinephrine (1 microgram/ml), sodium arachidonate (0.83 mM), thrombin (0.125 U/ml), and A-23187 (10 microM). The IC50 value (concentration producing 50% inhibition of aggregation) of W-7 was lower in arachidonate- and collagen-induced aggregation than in ADP- or epinephrine-induced aggregation. A good correlation between the potency in inhibition of collagen-induced aggregation by W-7 and its derivatives and their affinities to calmodulin was obtained (r = 0.94). Thus, the inhibitory mechanism of these compounds may be due to their effect on Ca2+-calmodulin-dependent processes, such as 20,000-dalton myosin light chain phosphorylation. These data also support the hypothesis that the calmodulin-mediated system has an important role in platelet function.

The Relationship Between the Hemorrhagic and Antithrombotic Properties of Low Molecular Weight Heparin in Rabbits

We have compared the hemorrhagic and antithrombotic effects of a low molecular weight (LMW) heparin fraction and standard heparin in rabbits. Similar LMW heparin fractions have antithrombotic effects when tested in animals, but their hemorrhagic effects relative to standard heparin have not been established. Standard porcine mucosal heparin (mol wt 15,000 daltons) was depolymerized by nitrous acid to a low molecular weight fraction (mol wt 4600 daltons). Using equal USP units, the standard and Dep LMW heparin were compared in vitro, ex vivo, and in vivo. In vitro, when diluted in rabbit plasma, the Dep LMW heparin at equivalent anti-Xa activity showed less prolongation of thrombin clotting times or activated partial thromboplastin times. Ex vivo, platelets from rabbits treated with the Dep LMW heparin showed less inhibition of collagen-induced aggregation. The relative hemorrhagic properties of the two heparins were compared in vivo in rabbits using a sensitive blood loss assay, and the antithrombotic properties were compared in a thrombin-induced venous stasis model. By using an optimal threshold heparin dose in each test system, it was possible to demonstrate that equal USP units of Dep LMW heparin caused less blood loss but showed greater antithrombotic activity than standard heparin.

The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits

We have compared the hemorrhagic and antithrombotic effects of a low molecular weight (LMW) heparin fraction and standard heparin in rabbits. Similar LMW heparin fractions have antithrombotic effects when tested in animals, but their hemorrhagic effects relative to standard heparin have not been established. Standard porcine mucosal heparin (mol wt 15,000 daltons) was depolymerized by nitrous acid to a low molecular weight fraction (mol wt 4600 daltons). Using equal USP units, the standard and Dep LMW heparin were compared in vitro, ex vivo, and in vivo. In vitro, when diluted in rabbit plasma, the Dep LMW heparin at equivalent anti-Xa activity showed less prolongation of thrombin clotting times or activated partial thromboplastin times. Ex vivo, platelets from rabbits treated with the Dep LMW heparin showed less inhibition of collagen-induced aggregation. The relative hemorrhagic properties of the two heparins were compared in vivo in rabbits using a sensitive blood loss assay, and the antithrombotic properties were compared in a thrombin-induced venous stasis model. By using an optimal threshold heparin dose in each test system, it was possible to demonstrate that equal USP units of Dep LMW heparin caused less blood loss but showed greater antithrombotic activity than standard heparin.

Some Characteristics of Mouse Platelet Aggregation and a Comparison of the Activity of a Range of Compounds in Mouse and Human Platelet-Rich Plasma in Vitro

Collegan-induced platelet aggregation in mouse citrated platelet-rich (PRP) was associated with 5-hydroxytryptamine (5-HT) release and degranulation. Adenosine 5'-diphosphate (ADP) induced monophasic and reversible aggregation with no degranulation. Mouse platelets gave no response to adrenaline but changed shape and sometimes aggregated in response to 5-HT. Both amines potentiated the effect of ADP. The respective potencies of prostaglandin E1, papaverine, VK 774, BL 3459 and adenosine as inhibitors of ADP-induced aggregation were similar in mous and human PRP. Although ticlopidine had similar activity in the two species, aspirin and flurbiprofen were considerably less potent in mouse than human PRP as inhibitors of collagen-induced aggregation. It is suggested that collagen-induced aggregation of mouse platelets in vitro occurs by a mechanism largely independent of arachidonic acid metabolites.

Fenbufen and biphenylacetic acid inhibit platelet function and the arachidonate prostaglandin system

Biphenylacetic acid or BPAA ([1,1-biphenyl]-4-acetic acid) is a metabolite of the non-steroidal anti-inflammatory drug, fenbufen (γ-oxo[1,1-biphenyl]-4-butanoic acid). A 0.12 mM concentration of BPAA produces almost complete inhibition of arachidonate-induced aggregation in vitro comparable to the effect produced by similar concentration of other non-steroidal anti-inflammatory agents. Fenbufen does not inhibit arachidonate-induced aggregation in vitro . These data correlate with the ability of BPAA, but not fenbufen, to inhibit platelet prostaglandin synthetase in vitro . Respiratory distress and histologic evidence of platelet aggregates formed in the pulmonary microvasculature following arachidonate infusion are markedly inhibited by oral doses of both fenbufen and BPAA. The order of efficacy (at 10 mg/kg) for inhibition of respiratory distress is BPAA > fenbufen > aspirin. These data confirm other studies indicating metabolic conversion of fenbufen in vivo to the active metabolite, BPAA. Collagen-induced aggregation is reduced by in vitro addition of either BPAA or fenbufen and in platelet-rich plasma from rats receiving oral doses of these compounds. BPAA added in vitro is more potent than either aspirin or fenbufen as an inhibitor of collagen-induced aggregation. Following in vivo dosing at 10 mg/kg, BPAA is comparable to aspirin in efficacy as an inhibitor of collagen-induced aggregation, while the same dose of fenbufen is less effective. The ability of BPAA to inhibit platelet aggregation appears to be related to intererence with the arachidonate-thromboxane-prostaglandin system. Fenbufen's inhibitory action may be dependent on conversion to BPAA or involve another mechanism. Other data suggest, however, that this mechanism is not related to inhibition of serotonin release or inhibition of phosphodiesterase activity in platelets.

Effect of orally administered sulphinpyrazone (Anturan R) on platelet aggregation in vitro

Platelet-aggregation tests were performed with citrated blood samples obtained from six healthy volunteers who had received,for investigational purposes, the unusually high single oral dose of 800 mg sulphinpyrazone, or placebo. Sulphinpyrazone inhibited collagen- and ADP-induced, but not adrenaline-induced aggregation. The maximum effect was observed after 2–4 hours, and in the case of collagen-induced aggregation the degree of inhibition was 39%. Sulphinpyrazone increased disaggregation after induction of aggregation with ADP. There was a significant correlation between the inhibition of collagen-induced aggregation and the concentration of sulphinpyrazone in the plasma. Plasma uric-acid levels were markedly decreased after treatment with sulphinpyrazone, but this effect was unrelated to the inhibition of aggregation.

Bleeding time in Uremia: A useful test to assess clinical bleeding

Modified Ivy bleeding time (template) and platelet aggregation to ADP, epinephrine, and collagen were studied in 26 uremic patients who had not recently ingested anti-platelet drugs. Regardless of the aggregating agent used, the abnormalities in platelet aggregation were often mild, even with advanced uremia, and frequently less severe than the effects of common anti-platelet drugs. The inhibition of collagen-induced aggregation was significantly correlated with both increased bleeding time and blood urea nitrogen. Platelet aggregation was not discriminative between clinically bleeding and non-bleeding groups of patients, but the bleeding time was helpful in this regard. In certain cases, the aggregometric patterns differed between drug-induced and uremic thrombocytopathies. Platelet aggregometry appears to be of little help clinically in assessing the severity of the uremic bleeding diathesis.