SummaryThe aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 μg/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 μM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 μg/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained twodimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on Ilia), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

The aim of this study was to investigate the platelets of a patient having bleeding episodes that began in infancy. The patient’s platelets in citrated-PRP did not aggregate when stimulated with ADP (5 and 10 uM), collagen (2.5 ug/ml), or sodium arachidonate (1 uM). However, washed patient platelets, in the presence of 2mM calcium, aggregated and secreted when stimulated with high concentrations of thrombin (0.36, 0.72 and lU/ml) or collagen (2, 4, 10 ug/ml). Monoclonal antibodies (Mab) LYP18 (directed against the IIb-IIIa glycoprotein complex) and LYP8 (anti-thrombospondin) inhibited thrombin and collagen induced aggregation of control but not the patient platelets. Patient thrombin -stimulated platelets did not bind 125I-labelled fibrinogen (40 to 320 ug/ml). Moreover, stimulating the washed patient's platelets with ADP (10-100 uM), in the presence of fibrinogen (2mg/ml), did not result in aggregation. Binding studies using Mab 125I-LYP2 (directed against the IIb-IIIa glycoprotein complex) showed the absence of the complex on the patient's platelets. The absence of the IIb-IIIa complex on the patient's platelets was also observed using crossed immunoelectro -phoresis and Mab 125I-LYP2 or 125I-LYP18. Individual glycoproteins (lib or Ilia) were not detected on silver stained two-dimensional (non-reduced/reduced) SDS-PAGE. Moreover, Western blots of |he patients platelets used in combination with anti-PLA or anti-LEK polyclonal antibodies failed to detect the presence of these two glycoproteins. These results indicate that this patient has Glanzmann's thrombasthenia or a variant of this disease. Moreover, this study shows that platelets lacking the IIb-IIIa glycoprotein complex can aggregate in responseto collagen or thrombin in the presence of physiological concentrations of calcium.

No abstract

Extracts of human subcutaneous tissue with a very low thromboplastic activity strongly stimulate platelets (sphering and pseudopode formation) within seconds if added to blood directly at blood sampling (30 μ/ml citrate blood). To study the effect of this hemostasis activating factor (HaF) on platelet retention a special glass bead column with basically low retention (0-15 % in healthy individuals) was prepared. If HaF 50μl/ml) was added to freshly drawn citrate blood retention was increased from a mean of 12 % to a mean of 57 %. Even higher retention rates were obtained by adding a cryoprecipitate from normal PPP (50μl/ml) together with HaF. 100μl/ml of cryoprecipitate markedly enhanced platelet retention. FeibaR (Immuno) alone did not increase platelet retention. Together with HaF FeibaR (50μl/ml citrate blood) induced a 85% retention. The enhancing effect of HaF on platelet retention is probably caused by rapid platelet stimulation and may be an essential trigger mechanism for primary hemostasis.