Decreased Arteriolar Endothelium-derived Relaxing Factor Production During the Development of Genetic Hypertension

In the present study, basal and stimulated production of endothelium-derived relaxing factor (EDRF) were examined in young (4—5 week-old) and mature (10—12 week-old) spontaneously hypertensive rats (SHR) and were compared with those of age-matched Wistar Kyoto (WKY) normotensive rats. N^w-nitro-L-arginine methyl ester (L-NAME) was used as a probe for basal EDRF production in vivo. We also determined the dose-response effects of acetylcholine (Ach, 10^?9-10^?5 M), sodium nitroprusside (NP, 10^?9-10^?5 M), and norepinephrine (NE)     

Factor VIII and Human Platelet Aggregation

SUMMARY Bovine factor VIII is a potent inducer of aggregation of human platelets. Upon gel filtration of five-thousand-fold purified material in 0.5 M CaCl₂, bovine factor VIII is separated into high and low molecular weight components: the former contains both a 'carrier protein'and platelet aggregating activity, the latter theproco-agulant activity (low molecular weight factor VHI, LMW-F VIII). Upon removal of Ca²⁺ ions, LMW-F VIII recombines with the 'carrier protein'. LMW-F VIII modifies aggregation by 'carrier protein', but not aggregation by undissociated bovine factor VIII, adenosine-5'-diphosphate or adrenaline. This finding indicates that the platelet aggregating activity is indeed a property of the 'carrier protein'.     

Factor VIII and Human Platelet Aggregation

During digestion of highly purified bovine factor VIII or neuraminidase-treated human factor VIII by plasmin the procoagulant activity is destroyed more rapidly than the aggregating activity. At an intermediate stage, fragments are transiently formed, which inhibit platelet aggregation by the respective undigested materials, but without corssed inhibition. During proteolysis by plasmin, human factor VIII retains antigenic and restocetin cofactor properties. Contrary to previous observations obtained with less purified preparations, plasmin digest of human or bovine factor VIII do not inhibit ADP-induced platelet aggregation. Thrombin and reptilase do not modify the aggragating activities of bovine and neuraminidase-treated human factor VIII.     

Platelet Aggregation and the Availability of Platelet Factor 3

W e have previously shown (Hardisty and Hutton, 1965, 1966) that kaolin makes platelet factor 3 (PF₃) available in platelet-rich plasma (PRP) by a reaction which involves platelet aggregation and which is inhibited by antagonisis of adenosine diphosphate (ADP), such as adenosine and related compounds. Using the kaolin clotting time system, we were not able to detect a significant effect of ADP on PRP in the absence of kaolin, and we therefore concluded that adhesion of platelets to an activating surface was also necessary for optimal effect, and that ADP aggregation alone did not make PF₃ available. This conclusion was at variance with those of Mustard, Hegardt, Rowsell and MacMillan (1964) and Castaldi, Larrieu and Caen (1965), who used different clotting systems and found evidence of increased clotting activity in platelets after ADP aggregation. We therefore decided to reinvestigate the effect of platelet aggregation on PF₃ availability, using the 'Stypven time' in order to eliminate the need for simultaneous activation of the earlier stages of the intrinsic coagulation process. Spaet and Cintron (1965) have already used this system to demonstrate the effect of kaolin and connective tissue on PF₃ availability.     

Platelet Aggregation and the Availability of Platelet Factor 3

We have previously shown (Hardisty and Hutton, 1965, 1966) that kaolin makes platelet factor 3 (PF₃) available in platelet-rich plasma (PRP) by a reaction which involves platelet aggregation and which is inhibited by antagonisis of adenosine diphosphate (ADP), such as adenosine and related compounds. Using the kaolin clotting time system, we were not able to detect a significant effect of ADP on PRP in the absence of kaolin, and we therefore concluded that adhesion of platelets to an activating surface was also necessary for optimal effect, and that ADP aggregation alone did not make PF₃ available. This conclusion was at variance with those of Mustard, Hegardt, Rowsell and MacMillan (1964) and Castaldi, Larrieu and Caen (1965), who used different clotting systems and found evidence of increased clotting activity in platelets after ADP aggregation. We therefore decided to reinvestigate the effect of platelet aggregation on PF₃ availability, using the ‘Stypven time’ in order to eliminate the need for simultaneous activation of the earlier stages of the intrinsic coagulation process. Spaet and Cintron (1965) have already used this system to demonstrate the effect of kaolin and connective tissue on PF₃ availability.     

Fish Oil Supplementation Inhibits Platelet Aggregation and ATP Release Induced by Platelet-activating Factor and Other Agonists

The anti-inflammatory effects of fish oil may partly be due to the inhibition of platelet activation induced by platelet-activating factor (PAF) and other agonists. To investigate this hypothesis, the diets of 12 healthy volunteers were supplemented with 12 fish oil capsules or 12 olive oil capsules daily for 4 weeks in a double blind crossover study. Aggregation induced by PAF (18 and 12.5 nM) and collagen (20 μg/ml)tended to be reduced after fish oil but the effect was statistically significant only in subjects receiving fish oil in the 6rst 4 weeks of the study (P 0.05, n=6). The effect of fish oil supplementation on platelet ATP release was more marked with significant inhibition of ATP release induced by PAF (1200 and 36 nM, P 0.01, n = 12), collagen (20 μg/ml, P 0.005, n = 12) and ADP (15,10 and 5 μM, P 0.05, n = 12). Olive oil supplementation appeared to inhibit ATP release induced by collagen (45 and 30 μg/ml, P> 0.025, n = 12), while aggregation and ATP release induced by arachidonic acid and adrenaline were unaffected by the supplements. Plasma fibrinogen was significantly reduced after olive oil (P 0.01, n = 12) while prothrombin time was reduced after fish oil (P 0.001, n = 12) and olive oil (P 0.0025). Reduced platelet aggregation and more importantly, inhibition of platelet release induced by PAF and other agonists may contribute to the anti-inflammatory effects of fish oil supplementation in a number of disease states but olive oil may also independently affect platelet function and influence the effect offish oil.     

Subcutaneous Selatogrel Inhibits Platelet Aggregation in Patients With Acute Myocardial Infarction

BackgroundOral P2Y₁₂ receptor antagonists exhibit delayed onset of platelet inhibition in patients with acute myocardial infarction (AMI). Selatogrel is a potent, highly selective, and reversible P2Y₁₂ receptor antagonist with a rapid onset and short duration of action.ObjectivesThis study sought to assess inhibition of platelet aggregation following subcutaneous administration of selatogrel in patients with AMI.MethodsPatients with AMI were randomized to a single subcutaneous dose of selatogrel of 8 or 16 mg. The primary endpoint was response to treatment (P2Y₁₂ reaction units <100; measured by VerifyNow) at 30 min post-dose. Safety was assessed up to 48 h post-injection.ResultsForty-seven patients received selatogrel 8 mg (n = 24) or 16 mg (n = 23) followed by ticagrelor (n = 43) or clopidogrel (n = 1). The proportion of responders 30 min post-dose was 91% (one-sided 97.5% confidence interval [CI]: 80% to 100%) and 96% (97.5% CI: 87% to 100%) with 8 and 16 mg, respectively (p values for responders >85% target; p = 0.142 and p = 0.009, respectively). Response rates were independent from type of AMI presentation, age, or sex. A similar response rate was observed at 15 min (8 mg: 75% [97.5% CI: 58% to 100%]; 16 mg: 91% [97.5% CI: 80% to 100%]), which was sustained at 60 min post-dose (8 mg: 75% [97.5% CI: 58% to 100%]; 16 mg: 96% [97.5% CI: 87% to 100%]). At 15 min, median P2Y₁₂ reaction units was 51 (range: 4 to 208) for 8 mg and 9 (range: 2 to 175) for 16 mg. Selatogrel was well tolerated, without major bleeding complications.ConclusionsSingle-dose subcutaneous administration of selatogrel in patients with AMI was safe and induced a profound, rapid, and dose-related antiplatelet response. (A Medical Research Study to Evaluate the Effects of ACT-246475 in Adults With Heart Attack; NCT03487445, 2018-000765-36 [EudraCT])     

Shear Rate Dependent Inhibition of Platelet Adhesion and Aggregation on Collagenous Surfaces by Antibodies to Human Factor VIII/von Willebrand Factor

The effect of heterologous and homologous antibodies to factor VIII/von Willebrand factor (F.VIII/VWF) and purified VWF on the interaction of human platelets with subendothelium and with a surface consisting of collagen fibrils was investigated using annular flow chambers. Wall shear rates of 200–5200 s⁻¹ were produced by varying the flow rate of the citrated blood which was circulated through these chambers by a pump. Surface coverage with platelets and with platelet aggregates was measured morphometrically. The antibodies had no effect on platelet adhesion at low shear rates. However, an increasing inhibition of adhesion was observed with increasing shear rates. The antibodies to F.VIII/VWF and to purified VWF virtually abolished adhesion at shear rates which are present in the microvasculature (1300–5200 s⁻¹). By contrast, antibodies to factor VIII:C which were isolated from two patients with haemophilia A had no effect on adhesion. In addition antibodies to F.VIII/VWF and to purified VWF inhibited adhesion-induced aggregation. We conclude that F.VIII/VWF plays an essential role as cofactor for platelet adhesion to subendothelium and collagen fibrils at high blood shear rates, i.e. when the time available for the establishment of a stable bond between platelets and collagenous surfaces is short.     

Inhibition of Endotoxic Lipopolysaccharide-mediated Platelet Aggregation by Cobra Venom Anticomplementary Factor

Aggregometry studies on endotoxic lipopolysaccharide (LPS)-mediated rabbitplatelet aggregation were performed.Different preparations of LPS showed characteristic aggregometry profiles, and LPSwith potent anticomplementary activitiesgenerally had a more vigorous plateletaggregation function than did LPS preparations with lesser anticomplementaryfunctions. Cobra venom anticomplementary factor (CVF) inhibited LPS-platelet interaction, and the inhibition wasboth time and dose dependent. Dose-response curves of CVF inhibition on LPSor zymosan-mediated platelet aggregation were essentially identical. In vitroand in vivo studies showed that CVFinhibition persisted even when hemolyticcomplement activities reached more than70% of those originally present. At thecritical time of days 5 or 6 following CVFadministration, the lack of platelet responses towards LPS could be restored byaddition of fresh plasma from normal orC6-deficient rabbits, but not with plasmathat had been treated with antigen—antibody complexes, zymosan, or heatingat 56° for 30 min. The experimental dataindicate that serum protein(s) other thanthe terminal complement components areinvolved in LPS-platelet interaction. Itseems most likely that the factor(s) perturbed reside in the mechanisms involvedin activation of the alternate pathway.Furthermore, it appears quite possible thatLPS-platelet interactions can be inhibitedby manipulating the humoral factor(s)involved rather than by altering the platelets themselves.

Submitted on January 9, 1974 Accepted on February 11, 1974     

Inhibition of Human Platelet Aggregation by Plasmin Digests of Human Factor VIII

Human factor VIII, upon prolonged incubation at 37°C with plasmin, inhibits plateletaggregation induced by ADP, adrenaline,or collagen. The human factor VIII digestsare not anticoagulant in the thrombin timeor partial thromboplastin time. Factor VIIIsplit products may interfere in vivo withplatelet function and thus could contributein some way to the hemorrhagic diathesisof fibrinolytic states.

Submitted on March 19, 1973 Revised on May 7, 1973 Accepted on May 11, 1973     

Influence of the Pure Synthetic PAF (Platelet Aggrregating Factor) on Clot Retraction and Platelet Aggregation

Pure synthetic platelet aggregating factor (PAF) (1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine) induces a dose-dependent platelet aggregation in platelet-rich plasma (PRP) and in gel-filtered platelets. Irreversible platelet aggregation was observed at final concentrations of PAF higher than 2 times 10^-7 mol/l, while reversible or two-wave aggregation was obtained with lower final concentrations. The second wave was inhibited by acetylsalicylic acid, indomethacin, dipyridamole, EDTA, EGTA, theophylline, caffeine, PGE₁ and verapamil. PAF does not induce reptilase clot retraction (RCR); however, it does not inhibit RCR induced by ADP or thrombin. Since all substances known to activate platelets also induce RCR, the lack of this activity by PAF would support the existence of a third pathway in platelets.     

Bivalirudin Inhibits Periprocedural Platelet Function and Tissue Factor Expression of Human Smooth Muscle Cells

Aim: A major concern of stent implantation after percutaneous coronary intervention (PCI) is acute stent thrombosis. Effective inhibition of periprocedural platelet function in patients with coronary artery disease (CAD) leads to an improved outcome. In this study, we examined the periprocedural platelet reactivity after administrating bivalirudin during PCI compared to unfractionated heparin (UFH) administration. Further, the effect of bivalirudin on induced tissue factor (TF) expression in smooth muscle cells (SMC) was determined. Methods: Patients with CAD (n = 58) and double antithrombotic medication were treated intraprocedural with UFH (n = 30) or bivalirudin (n = 28). Platelet activation markers were flow cytometrically measured before and after stenting. The expression of TF in SMC was determined by real‐time PCR and Western blotting. The thrombogenicity of platelet‐derived microparticles and SMC was assessed via a TF activity assay. Results: Bivalirudin significantly diminished the agonist‐induced platelet reactivity post‐PCI. Compared to UFH treatment, the adenosine diphosphate (ADP) and thrombin receptor‐activating peptide (TRAP)‐induced thrombospondin expression post‐PCI was reduced when bivalirudin was administrated during intervention. In contrast to UFH, bivalirudin reduced the P‐selectin expression of unstimulated and ADP‐induced platelets post‐PCI. Moreover, bivalirudin inhibited the thrombin‐, but not FVIIa‐ or FVIIa/FX‐induced TF expression and pro‐coagulant TF activity of SMC. Moreover, bivalirudin reduced the TF activity of platelet‐derived microparticles postinduction with TRAP or ADP. Conclusions: Bivalirudin is better than UFH in reducing periprocedural platelet activation. Moreover, thrombin‐induced TF expression is inhibited by bivalirudin. Thus, bivalirudin seems to be a better anticoagulant during PCI than UFH.     

A Platelet Defect Characterised by Absent Adrenaline-induced Aggregation and Lack of Secondary Aggregation in Response to ADP and Platelet-activating Factor

We describe a platelet defect characterised by absence of aggregation to adrenaline and lack of secondary aggregation to ADP and platelet-activating factor (PAF) in a male subject without a significant bleeding tendency. The platelets also exhibited a decreased response to the phorbol ester PMA and to vasopressin polypeptides. We obtained evidence that the defect did not involve the cyclooxygenase pathway and that it was not dependent on altered intraplatelet levels of cyclic AMP (cAMP). Furthermore, adrenaline-stimulated cAMP production was normal. Irreversible aggregation was obtained in response to a combination of two stimuli at subaggregating concentrations. This observation indicates that the lack of a bleeding tendency in this subject may be ascribed to subthreshold concentrations of agonists in vivo overcoming the defect to a single agonist.     

Different Requirements for Intrinsic Factor-Xa Forming Activity and Platelet Factor 3 Activity and their Relationship to Platelet Aggregation and Secretion

Summary . Platelets provide coagulant activity in part by promoting two essential reactions of intrinsic coagulation: (I) factor-X activation by a complex of factor IXa, factor VIII, calcium and platelets (intrinsic factor-Xa forming activity or XaFA); and (2) prothrombin activation by a complex of factor Xa, factor V, calcium and platelets (platelet factor 3 activity or PF₃A). We found that a chloroform extract of acetone-dried brain had about half the XaFA of a petroleum ether extract of brain and over twice the PF₃A of the petroleum ether extract; and that when purified phospholipids other than phosphatidylserine (PS) were added to PS, the resultant activity, compared with PS alone, was reduced in the assay for XaFA and enhanced in the assay for PF₃A. In experiments with washed platelets stirred with collagen XaFA developed rapidly (100% activity at 30 s), well before maximum [¹⁴C]5HT release (2-4 min) and decayed to 15% at 10 min. In contrast, PF₃A developed slowly (100% activity after 20 min) well after maximal aggregation and release. Maximal XaFA developed in unstirred platelet suspensions incubated with collagen, whereas PF₃A did not become available unless platelet suspensions were stirred and underwent secretion. Platelets stirred with ADP released [¹⁴C]5HT and developed PF₃A but not XaFA. Both [¹⁴C]5HT release and PF₃A by collagen were inhibited by p-chloromercuriphenylsulphonate, indomethacin and a combination of prostaglandin E₁ and RA 233, but XaFA was only minimally affected by these inhibitors. In contrast concanavalin A inhibited collagen-induced PF₃A and XaFA in dose-dependent fashion but had no effect on [¹⁴C]5HT release. Both platelet coagulant activities and [¹⁴C]5HT release were inhibited by aspirin, EDTA and a combination of antimycin A and 2-deoxy-D-glucose. These results indicate that the biochemical determinants of XaFA and PF₃A are different. PF₃A developed only when aggregation and release occurred whereas XaFA was independent of aggregation and release.     

Studies on the Mechanism of Ristocetin-induced Platelet Aggregation: Binding of Factor VIII to Platelets

The effect of ristocetin on the binding of [125I]factor VIII to platelets was studied. High and low affinity F.VIII binding sites exist on platelets. The high affinity sites bind 13 times more F.VIII than the low affinity sites. Ristocetin increased the binding of F.VIII to both types of binding sites by increasing the affinity of F.VIII for the platelet and increasing the total number of platelet binding sites. Chymotrypsin-treated platelets were not aggregated by ristocetin and F.VIII: these platelets have less of the major platelet membrane glycoproteins and bind much less [125I]F.VIII than do buffer-treated platelets with and without ristocetin.     

Racial Differences in Ristocetin-induced Platelet Aggregation

S ummary . Several investigators have reported defective ristocetin-induced platelet aggregation (RIPA) in individuals whose red blood cells contain sickle haemoglobin, but the race of control subjects in these studies was not stated. Therefore, maximal amplitude of RIPA was examined in 75 normal whites and blacks, 16 of whom had sickle trait defined by haemoglobin electrophoresis and sickle prep. Final ristocetin concentrations in platelet rich plasma were 1·1,1·2 and 1·5 mg/ml. Mean aggregation at 1·1 mg/ml was significantly less in blacks (mean 31%) than in whites (mean 72%) ( P < 0·001). 60% of blacks but only 11% of whites had less than 50% RIPA at 1·1 mg/ml. RIPA was entirely absent in 19% of blacks. Differences in RIPA between black and white subjects were also present at ristocetin concentrations of 1·2 and 1·5 mg/ml but were less striking. RIPA in 25 children with homozygous sickle cell anaemia was similar to that in the normal AA and AS blacks. Differences in RIPA could not be explained by age, sex, presence of sickle haemoglobin, or medications. Addition of normal plasma or platelets did not correct reduced RIPA in seven blacks, and their plasma inhibited normal RIPA responses.
Reduced platelet aggregation to low concentrations of ristocetin is a normal finding in many blacks, is not related to the presence of sickle haemoglobin, and appears to be due to a plasma inhibitor against RIPA.     

Platelet Aggregation in Neonates with Hyperbilirubinaemia

In 19 jaundiced newborns who had no evidence of kernicterus the effect of bilirubin on adenosine-5′-diphosphate (ADP), epinephrine and collagen-induced platelet aggregation was studied. Compared to 20 normal adult controls, no significant difference was found for ADP and collagen-induced platelet aggregation in jaundiced infants. However, with epinephrine, significantly decreased aggregation was observed, but this was also the case for non-jaundiced infants. This suggests that the reduced response to epinephrine in hyperbilirubinaemic infants is related to age rather than to bilirubin. Increased bilirubin is therefore not responsible for any increased bleeding tendency due to impaired platelet function.     

Effects of Recombinant Human Megakaryocyte Growth and Development Factor (rHuMGDF) on Platelet Production,Platelet Aggregation,and Thrombosis

Recombinant human megakaryocyte growth and development factor (rHuMGDF) is a c-mpl ligand that promotes the differentiation of CD34+ precursor cells into megakaryocytes and then platelets. In experimental animals, injection of this and other c-mpl ligands leads to profound increases in the circulating platelet count in a matter of days. However, c-mpl ligands have also been shown to sensitize platelets to aggregating agents in vitro, raising the possibility that c-mpl ligands may have prothrombotic effects in vivo. Therefore, characterizing rHuMGDF in an in vivo model of thrombosis is a necessary and critical step in defining the in vivo pharmacology of this novel and important hematopoietic factor, a pegylated form of which is currently in clinical trials. To determine the biologically effective doses in the rabbit, daily subcutaneous injections of rHuMGDF at 0.1, 1.0, or 10 µg/kg were administered over 7 days. Daily injection of 10 µg/kg produced an approximate fourfold increase in platelet count and 1.0 µg/kg doubled platelet count over the injection period, both of which were statistically significant. The serum concentrations of rHuMGDF were determined 10 minutes following a single intravenous injection with 0.1, 1.0, and 10 µg/kg, and were 0.05 ± 0.02, 0.98 ± 0.07, and 21.32 ± 2.35 ng/ml. To determine whether rHuMGDF can sensitize platelets in vivo, platelet aggregometry was performed on platelets isolated from animals immediately before and 10 minutes after they had been injected intravenously with rHuMGDF (0.1, 1.0, and 10 µg/kg). Intravenous injection of 10 µg/kg produced measurable changes in platelet aggregometry ex vivo, as evidenced by an increased sensitivity of platelets to adenosine diphosphate (ADP). To assess the in vivo prothrombotic potential of rHuMGDF, a rabbit carotid artery model of cyclic flow reduction (CFR) was used to measure the effect of intravenous rHuMGDF administration on the rate of thrombus formation as assessed by CFR slope and frequency. Intravenous administration of rHuMGDF had no effect on CFR slope or frequency when administered in doses ranging from 0.1 to 10 µg/kg. Control experiments demonstrated that CFR slope and frequency can be enhanced by intravenous infusion of epinephrine and can be abolished by the combined administration of aspirin and ketanserin, indicating that potentially prothrombotic and antithrombotic agents can be identified in this model. We conclude that biologically active doses of rHuMGDF used in this study (1.0 and 10 µg/kg) produce measurable serum levels, induce a thrombopoietic effect, and sensitize platelets in vivo, as determined by ex vivo aggregometry, at 10 µg/kg. Despite the sensitization of platelets to aggregation induced by ADP, it is clear that rHuMGDF does not alter the pattern of CFRs observed in the rabbit carotid artery, whereas agents known to sensitize platelets (epinephrine) and to inhibit platelets (aspirin and ketanserin) readily affected the CFR pattern. These findings indicate that intravenous rHuMGDF administration, while capable of sensitizing platelets, does not enhance platelet-dependent thrombosis in vivo.