Significance of platelet activation in sickle cell anaemia.

BACKGROUND: There is increasing evidence suggesting the contribution of platelets in the vaso-occlusive phenomena found in sickle cell anaemia. This study is aimed at using simple, inexpensive parameters to determine the role of platelets in the steady state and vaso-occlusive crisis state of Nigerian sickle cell anaemia patients. METHODS: The circulating platelet aggregate (CPA) ratio, platelet factor-3 availability (PF-3) and platelet counts of 60 adult Nigerian sickle cell anaemia patients were studied. RESULTS: The CPA ratio in the sickle cell anaemia (SCA) patients in steady state (SS) was 0.93 +/- 0.05, 0.89 +/- 0.04 during vaso-occlusive crisis (VOC) and 0.98 +/- 0.02 in the control group (C). The values in the vaso-occlusive crisis and in steady state were significantly lower than in the control group (P < 0.05). PF3 availability in steady state and vaso-occlusive crisis were 29.7 +/- 4.0 secs and 28.4 +/- secs respectively. The times are significantly shorter when compared with the control group with a time of 36.2 +/- 4.3 secs (P < 0.05). There was however no significant difference between the two sickle cell groups. Platelet count was significantly raised in the steady state patients 224.3 +/- 46.3 x 10(9)/L when compared with controls of 196.6 +/- 39.3 x 10(9)/L (P < 0.05). There was a significant fall during VOC to 140.6 +/- 36.3 x 10(9)/L (P < 0.05). The difference between the two sickle cell groups is significant (P < 0.05). CONCLUSION: This study indicates varying degrees of partial activation of platelets in vivo in the steady state and vaso-occlusive crisis state of sickle cell anaemia. It support's a contribution of platelet to the vascular occlusion that underlies much of the morbidity in the disease.

Haemarthroses after total knee arthroplasty caused by an isolated platelet factor 3 availability defect.

We present seven patients with recurrent haemarthroses after total knee arthroplasty, caused by an inherent platelet function defect. These patients developed painful knee swelling, persistent bleeding and/or wound breakdown, a platelet factor 3 availability defect being identified in all cases. Surgical exploration, with joint debridement, lavage and synovectomy, was performed in four patients who did not improve with conservative therapy. Histopathological examination of synovium revealed a focal synovial reaction with histiocytic infiltration, and occasional foreign-body giant cells. One patient required an early revision because of aseptic loosening of their tibial component. The condition was treated by single-donor platelet transfusions with good results. The diagnosis, management, and relevance of this disorder are discussed.

Association of factor V activity with membranous vesicles released from human platelets: requirement for platelet stimulation.

The membrane-associated factor V-like activity (platelet factor 1, PF1) and the phospholipid-like catalytic surface activity (platelet factor 3, PF3) were studied in human platelets from normal and two factor V-deficient donors. Collagen stimulation or mechanical disruption of gel-filtered platelets was necessary for the expression of significant amounts of PF1 and PF3. Stimulation was also necessary for the uptake of factor V or Va by PF1-deficient platelets from the factor V-deficient donors. The activity of PF1 was also generated by association of factor V or Va with membrane-rich fractions obtained by gel filtration of the supernatant from collagen-stimulated or frozen-thawed PF1-deficient platelets. The amount of PF1 obtained by such all-or-none binding experiments was directly proportional to the amount of PF3 already expressed in the platelet preparation. These data have been summarized in terms of a hypothesis which views PF1 and PF3 to be activities associated with membranous vesicles released from platelets only after stimulation.

Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3.

The relationship between the appearance of membrane-associated factor V-like activity (platelet factor 1, PF1) and phospholipid-like catalytic activity (platelet factor 3, PF3) has been examined, in vitro, in collagen-stimulated, human platelets. Both activities increased 7 fold upon collagen treatment relative to stirred controls. After sedimentation of stimulated platelets, 31% of total PF1 and 41% of PF3 remained in the supernatant fraction. PF1 eluted from a Sepharose CL-4B column in the same void volume fractions as PF3, phospholipid, and vesicular particles. These fractions had roughly 100 fold (lipid basis) or 1000 fold (protein basis) enhanced specific activity when compared to the stimulated platelet suspension. Freeze-fracture electron microscopy demonstrated that these void volume fractions contain two populations of membranous vesicles (80-200 nm and 400-600 nm in diameter). Upon centrifugation of the void volume fractions, PF1 and PF3 activities, phosphate-containing material, and ultraviolet-absorbing material all sedimented at the same rate, indicating that PF1 and PF3 are activities associated with one or both of the platelet-derived vesicle populations. Finally, we examined the effects of inhibitors on the appearance of PF1, PF3, platelet factor 4, total intrinsic factor V activity, and serotonin as well as on platelet aggregation. These studies suggest that the collagen-stimulated release of PF1 and PF3 is not coupled to either platelet aggregation or PF4 release but is probably a separate phase of the release reaction.

The role of phospholipids in platelet function.

Platelet phospholipids undergo significant alterations during aggregation induced by thrombin or other agents. There is an early increase in phosphatidic acid, with a decrease in phosphatidyl inositol. De novo synthesis of most phospholipids from 14C-glycerol is decreased. Thrombin stimulates 32P-phosphate incorporation into di- and triphosphoinositides, suggesting increased phosphorylation of phosphatidyl inositol during aggregation. Arachidonic acid for prostaglandin synthesis is released from platelet phospholipids. Thrombin induced aggregation results in release of arachidonic acid primarily from phosphatidyl choline and phosphatidyl inositol. The availability of free arachidonic acid may be regulated by platelet phospholipase A2 activity. The latter activity is stimulated by thrombin, requires calcium ions, and is inhibited by agents which elevate cyclic adenosine monophosphate. Phospholipids are probably an essential component of the platelet surface lipoprotein procoagulant activity known as platelet factor 3. There is evidence that calcium ions may mediate binding between gamma carboxyglutamic acid residues on the amino terminal portion of prothrombin and negatively charged phosphate groups on phospholipid micelles. Binding of prothrombin to phospholipid on the platelet surface may orient the former such as to facilitate the prothrombinase activity of Factor Xa. Platelet phospholipids and platelet factor 3 activity are decreased in some congenital and myeloproliferative disorders. Increases in these factors may be associated with thrombotic and arterial occlusive disorders.

PF3 activity in normal subjects and beta-thalassemia trait.

Platelet factor 3 (PF3) is a platelet membrane component that plays an important role in the activation of the coagulation mechanism. Whenever platelet activation occurred, PF3 is released and participates in thrombin formation. Erythrocyte membrane fraction has also some PF3 like activity, and in abnormal erythrocyte membrane disorders, eg thalassemia, some of the membrane fraction accelerates platelet activation by increasing the PF3 activity. Formerly it was difficult to measure the PF3 activity in plasma. Recently a sensitive chromogenic test to determine the PF3 activity, which could detect the changes in PF3 activity with time, was introduced. This study was done to observe the effect of abnormal erythrocyte on platelet activation. The results obtained using the chromogenic method are the following: whole blood taken from normal subjects showed OD 0.11 +/- 0.06 at 0 minutes after blood collection and then increased significantly (p < 0.01) to 0.21 +/- 0.10 after 90 minutes, while the platelet count did not differ significantly (p > 0.05). Those results showed that there were some platelet activation after 90 minutes as seen by the increased PF3 activity, with no significant change in platelet counts. In beta-thalassemic trait subjects the PF3 activity in whole blood at 0 minutes did not differ significantly compared to the normal subjects, but after 90 minutes it was significantly higher (p < 0.01), OD 0.52 +/- 0.35. However the PF3 in platelet rich plasma at 90 minutes did not increase. The platelet count after 90 minutes was significantly decreased (p < 0.01) This result suggest that the increase in PF3 activity was caused by the role of the abnormal erythrocytes.

Development of a new method for detection of platelet factor 3 like activity.

We asked the question, "Can thalassemic erythrocytes play some role in alteration of the hemostatic system?", because clinical examination of thalassemic patients shows symptoms and signs related to alterations in hemostatic and circulatory systems, and thalassemic erythrocytes are different from normal erythrocytes. We obtained one of the answers to the question: The erythrocytes of postsplenectomized patients of beta-thalassemia/HbE disease could stimulate their own platelets to aggregate spontaneously. To know the role of erythrocytes in platelet aggregation, we wanted to examine the effect of thalassemic erythrocytes on the coagulation system by focusing of PF3-like activity of erythrocytes, because PF3-like activity of the ghosts of erythrocytes had been reported. For the study, we tried to develop a technique that was accurate and sensitive enough to detect PF3-like activity of blood. The system we developed was the following: 1) We activated the intrinsic coagulation pathway of commercial standard plasma by ellagic acid. 2) CaCl2, a fixed amount of PF 3 and synthetic thrombin inhibitor MD 805 were added to the reaction mixture. 3) At a fixed time, thrombin activity in the mixture was measured by using S-2238 as a substrate. At full activation of the contact system by ellagic acid, the amount of thrombin formed in a certain time depended on the amount of PF3-like substances such as cephalin, freeze-thawed platelets or ghosts of erythrocytes added to the test system, indicating that PF3-like activity of those substances can be measured by the activity of thrombin generated in a fixed time.(ABSTRACT TRUNCATED AT 250 WORDS)