Contribution of the Platelet Factor V Content to Platelet Factor 3 Activity

S ummary . The procoagulant activity obtained from bovine thrombocytes has been compared to that of lipids isolated from platelets, with and without the addition of purified bovine factor V. A one-stage assay, which consisted of delipidated bovine plasma containing RVV-activated factor X, was used to assess the activity. At low lipid concentrations no difference in coagulant activity was found between sonicated vesicles of extracted platelet lipid and lysed platelets. At higher lipid concentrations, however, the extracted lipids were found to be less active than lysed platelets. Determination of factor V in suspensions of gel-filtered platelets demonstrated that suspensions containing 2 × 10⁹ platelets per ml possessed about 1% of the factor V activity present in a normal bovine plasma pool. Platelet lysis by sonication produced a five-fold increase in factor V activity. Addition of factor V to sonicated vesicles of extracted platelet lipid, so as to produce an identical factor V activity per amount of lipid as found in lysed platelets, decreased the clotting time only in the higher lipid concentration range. A further three-fold increase in the amount of factor V added to the lipid vesicles made the coagulant properties of the lipid vesicles indistinguishable from those of lysed platelets over the whole range of phospholipid concentrations tested. When the conditions of the test were changed by diminishing the concentration of factor Xa in the substrate plasma, the difference between lysed platelets and extracted platelet lipid disappeared completely. It is concluded that the higher coagulant activity of lysed platelets, as compared to that of extracted platelet lipid, can be ascribed to platelet factor V activity. Therefore there is no compelling necessity to postulate the existence of a specific procoagulant factor in the platelet other than factor V or phospholipids.     

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 a Factor Enhancing Colicin E3 Activity In Vitro

The mechanism of action of colicin E3 (E3) was investigated in an in vitro system. Purified ribosomes are less susceptible to E3 than crude or washed ribosomes. A factor was found in the supernatant fraction of normal Escherichia coli cells that stimulates inactivation of ribosomes by E3, and on addition of this factor, about one tenth as much E3 was required for inactivation of ribosomes. On heating a mixture of E3 and this factor above 60 degrees , the ribosome inactivating activity of E3 increased greatly, and an amount corresponding to 0.01 mug of E3 was sufficient to inactivate 1.0 A(260) unit of ribosomes completely. By this treatment bacteriocidal activity of E3 decreased considerably, as the ratio of the two activities of E3 (ribosome inactivating activity and bacteriocidal activity) increased to 6 x 10(4)-fold. It is evident that the two activities do not run in parallel.This heat-treated product cleaved 16S rRNA in the same way as E3. These results suggest that inactivation of ribosomes is not due to colicin molecules prepared by the standard procedure, but to a modified form of them.     

The Development of Platelet Factor 3 Activity by Latex Particles--An Interaction with Plasma

Platelet factor 3 activity of polystyrene latex particles was found to beabsent when the particles were tested without prior incubation with plasma.Following such incubation, the particles developed considerable but suboptimal activity. An interaction between the particles and some plasmacomponent(s) during the incubation period which gives rise to a labile typeof activity has been postulated.

Submitted on July 10, 1963 Accepted on August 6, 1963     

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.     

Analysis of Platelet Factor 3 in Platelet Concentrates Stored for Transfusion

The amount of platelet factor 3 (PF3) activity expressed in stored platelet concentrates (PC) was measured in conjunction with extracellular LDH levels. Standard manual techniques for preparation of PC resulted in PF3 and LDH levels remarkably higher than those observed in PC prepared by apheresis or special manual plateletpheresis. During storage of PC, PF3 activity rose 2- to 10-fold, while LDH levels rose less than 2-fold over starting values. Loss of LDH and appearance of PF3 expressed as a percent of total per platelet were significantly correlated only in standard, manual PC. Approximately half of the PF3 activity observed in any type of PC remained in the supernatant plasma after centrifugation. Upon gel filtration, the supernatant PF3 activity eluted in a high molecular weight peak containing phosphate and light-scattering material. Our findings indicate that platelets in standard, citrated PC express PF3 in amounts that approach that of frozen-thawed (lysed) platelets; however, the manner in which the PF3 activity appears suggests that stored platelets undergo a combination of activation and damage processes.     

Platelet Antiheparin Activity

S ummary . A method for assaying the heparin-neutralizing activity of platelets washed by albumin density gradient separation (ADGS) is described. Residual heparin, not neutralized by platelets, is measured by its capacity to potentiate the inactivation of factor Xa by antifactor Xa. The assay is sensitive to concentrations of heparin as low as 5 × 10⁻⁴ units (approximately 5 ng) per ml. One major advantage of the assay is that it specifically detects the heparin-neutralizing and not the clot-promoting effect of platelets. The calibration curve relating the logarithm of clotting time to heparin concentration is linear. Platelets washed by ADGS (original method) spontaneously released 35% of the total antiheparin activity, where as only 2.75% of the total antiheparin activity was spontaneously released from platelets washed by a modified method of ADGS. Normal platelets washed by ADGS (modified method) and solubilized with Triton X-100 neutralized 19.08 units of heparin per 10¹⁰ platelets (mean of 19 samples). Two patients with platelet adenine nucleotide 'storage pool deficiency' had 16.3% and 21.4% of normal total antiheparin activity.     

A Standardized Bioassay for Platelet Factor 3 Released by Kaolin

The test for platelet factor 3 described by Hardisty & Hutton (1975) has been modified to conform to the usual design for a parallel-line bioassay. Manchester Comparative Thromboplastin has been used as assay Standard, allowing an arbitrary unit of activity to be adopted. However, experiments suggested that the platelet activity measured was different from tissue factor activity. Platelets are tested as platelet-rich plasma diluted in a standardized mixture of plasma and fibrinogen, so that differences between the clotting factors of the test samples are eliminated, as verified by experiments on haemophiliacs and patients on anticoagulant treatment. Sonication and repeated freezing and thawing of platelet-rich plasma showed that approximately 15% of the PF3 is released by kaolin. In vivo, a single dose of 600 mg of aspirin reduced the PR3-release to half the previous value in 2 h; initial values were regained in 5-8 days.     

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.     

Arrhythmias Caused by Platelet Activating Factor

Platelet Activating Factor and Arrhythmias. Introduction: Both ischemia and reperfusion are associated with ventricular arrhythmias. In both instances, neutrophils migrate into the ischemic zone, are activated by locally released factors, and hind to myocytes. The activated neutrophils liberate platelet activating factor (PAF). We have studied the arrhythmogenic actions of PAF on transmembrane potentials of isolated canine cardiac myocytes. Methods and Results: Cardiac myocytes were prepared from normal canine hearts by standard methods and studied in vitro by recording transmembrane potentials under control conditions and during exposure to graded doses of PAF, usually 0.25 to 1.25 μg (0.25 to 1.2 μM). Myocytes were superfused with Tyrode's solution (2.0 mL/min), paced at a cycle length of 1000 msec, and maintained at a temperature between 36° and 38°C. PAF caused a consistent and dose-dependent set of alterations in the transmembrane potential, including increased action potential duration, runs of early afterdepolarizations (EADs), and transient arrest of repolarization (PA). In addition, in some myocytes PAF caused intermittent small depolarizations both at the plateau voltage and resting potential. The effects of PAF were transient: only some residual action potential prolongation was noted after Tyrode's washout for 5 minutes. Effects of PAF were blocked in a dose-dependent manner by the PAF receptor antagonist, CV-6209. Both tetrodotoxin (1.2× 10^-6 M)and xylocaine (5 × 10^-5 M) antagonized the ability of PAF to cause EADs and PA. Conclusions: PAF consistently exerts arrhythmogenic effects on the membrane of ventricular myocytes. Since PAF is liberated by activated neutrophils and since activated neutrophils migrate into ischemic myocardium on reperfusion, we judge that PAF liberated by such neutrophils is an important arrhythmogenic factor for reperfusion arrhythmias. The same mechanism may be a cause of arrhythmias during the evolution of infarction.     

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.     

Acquired von Willebrand Syndrome with Inhibitors Both to Factor VIII Clotting Activity and Ristocetin-Induced Platelet Aggregation

A case of acquired von Willebrand's syndrome (vWs) is described which appeared to be due to antibodies directed against factor VIII clotting activity (FVIIIC), factor VIII-related antigen (FVIIIRAg) and von Willebrand factor. The antibodies directed against FVIIIRAg was demonstrated by the inhibitory effect of a platelet eluate on Ristocetin-induced aggregation of normal platelets. This effect was not shown by the patient's platelet-poor plasma alone, nor could it be demonstrated in platelet eluates from 13 other patients who had antibodies to FVIIIC but in whom there was no evidence of an acquired vWs.     

Ⅱ期高血压高胰岛素血症与血小板功能

Ⅱ期高血压高胰岛素血症与血小板功能＊万军戴政德杨顺裕毛方方李银书（武汉市第五医院心内科４３００５０）ＨｙｐｅｒｉｎｓｕｌｉｅｍｉａａｎｄＰｌａｔｅｌｅｔＡｃｔｉｖｉｔｙｉｎＨｙｐｅｒｔｅｎｓｉｏｎＷａｎＪｕｎ，ＤａｉＺｈｅｎｇｄｅ，ＹａｎｇＳｈｕｎｙ...     

Immunologic Studies of Platelet Protein

Antigens were demonstrated in platelets which were not detectable in plasma. One of these antigens was detected in serum and was presumed to be released during the process of coagulation. Plasminogen and fibrinogen were detected in platelets, and the fibrinogen appears to be part of the intracellularstructure. Thrombin was found to act not only on fibrinogen but also on atleast one other unidentified platelet protein.

Submitted on April 10, 1964 Accepted on September 3, 1964