Jacques P. Caen

Immunochemical Evidence for Protein Abnormalities in Platelets from Patients with Glanzmann's Thrombasthenia and Bernard-Soulier Syndrome

Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed (125)I iodination of normal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two (125)I-labeled glycoprotein bands, which migrate as GP IIb and GP IIIa. SDS-PAGE of (125)I-labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP IIb and GP IIIa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity observed in the GP I region after SDS-PAGE is associated with protein 17 and not with glycocalicin.

Analysis of the glycoprotein and protein composition of Bernard-Soulier platelets by single and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Previous reports have described conflicting results concerning the glycoprotein (GP) and protein composition of Bernard-Soulier platelets. In view of this controversy we have analyzed the platelets of four Bernard-Soulier patients using improved single and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis procedures. An absence of staining for carbohydrate of membrane GP Ib was characteristic for the platelets of each patient. Major periodate-Schiff staining bands corresponding to membrane GP IIb, IIIa, and IIIb were clearly detected and their presence was confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis. The protein content of the Bernard-Soulier platelets was increased two- to fourfold. However, analysis of their protein composition using 7-12% acrylamide gradient gels showed normal polypeptide profiles. Lactoperoxidase-catalyzed 125I-labeling of the Bernard-Soulier platelet surface proteins was followed by SDS-polyacrylamide gel electrophoresis and autoradiography. No labeling in the Ib position was detected whereas the other major membrane GP, including Ia and IIa, were normally located. In contrast, GP Ib was clearly detected by periodate-Schiff staining and autoradiography when normal human platelets that had been exhaustively treated with neuraminidase before the lactoperoxidase-catalyzed iodination were analysed. No abnormalities were detected in the GP patterns of membranes isolated from the patients erythrocytes. Only a severe molecular abnormality or possible deletion of GP Ib could account for this major platelet lesion in the Bernard-Soulier syndrome.

Effect of arsenic trioxide on viability, proliferation, and apoptosis in human megakaryocytic leukemia cell lines

Arsenic trioxide (As2O3) has been demonstrated to be effective for the treatment of acute promyelocytic leukemia (APL) and to inhibit proliferation and produce apoptosis in the APL cell line NB4. To determine if As2O3 might be useful for the treatment of other lineages, we investigated the effects of As2O3 on viability, proliferation, and induction of apoptosis in the megakaryocytic leukemia cell lines HEL, Meg-01, UT7, and M07e. Our results showed that As2O3, at concentrations of 0.1-2.0 microM, causes a dose- and time-dependent inhibition of survival and growth in all four megakaryocytic leukemia cell lines studied. In contrast, As2O3 at similar concentrations had no effects on either viability or growth of the nonmegakaryocytic leukemia cell line HL60 and two human breast cancer cell lines, ZR75 and MCF7. In situ end-labeling of DNA fragments (TUNEL assay) indicated that As2O3, at concentrations of 0.5-2 microM, could significantly induce apoptosis in the aforementioned four megakaryocytic leukemia cell lines, but not in the nonmegakaryocytic HL60, ZR75, and MCF7 cell lines. These results were confirmed using conventional morphologic assessment and the DNA ladder assay. Induction of apoptosis in arsenic-treated Meg-01 and UT7 cells was accompanied by a dose-response decrease of Bcl-2 protein, whereas As2O3 had no effect on this measurement in HL60, ZR75, and MCF7 cell lines. Pertinently, these concentrations of As2O3 produced identical changes in the characteristics of the APL cell line NB4. Collectively, these data demonstrate that As2O3 can selectively inhibit growth and induce apoptosis in megakaryocytic leukemia cell lines. The use of As2O3 for the treatment of malignant megakaryocytic disorders should be considered.

Thromboxane Synthesis and the Platelet Release Reaction in Bernard-Soulier Syndrome, Thrombasthenia Glanzmann and Hermansky-Pudlak Syndrome

Thromboxane (TX) synthesis was investigated in some characterized platelet disorders and correlated with release of ADP or [14C]serotonin and platelet aggregation. In a case with Bernard‐Soulier syndrome TX‐synthesis as well as ADP‐[14C]serotonin release and platelet aggregation were found to be essentially normal when platelets were incubated with collagen, ADP, arachidonic acid or prostaglandin G2 (PGG2). In a case with Hermansky‐Pudlak syndrome aggregation was normal with ADP, whereas aggregation with collagen and PGG2 was markedly decreased. Release of ADP and [14C]serotonin was found to be decreased with all investigated inducers in this platelet disorder, whereas TX‐synthesis was normal with arachidonic acid and PGG2. In three cases with Glanzmanns thrombasthenia almost no aggregation was observed with any of these compounds. However, in this disorder platelet TX‐formation from arachidonic acid or PGG2 was also normal whereas ADP and collagen both induced less TX‐formation than in normal platelets.

Effect of κ-casein split peptides on platelet aggregation and on thrombus formation in the guinea-pig

An undecapeptide (residues 106-116 of cow kappa-casein) is known to inhibit human platelet aggregation and fibrinogen binding through inhibition of the interaction between the fibrinogen gamma-chain C-terminus and alphaIIbbeta3. This was due to structural homologies with the fibrinogen gamma-chain C-terminal dodecapeptide. We have therefore compared in this work the in vitro anti-aggregating activity of kappa-casein split peptides and their in vivo potential antithrombotic activity in a model of arterial thrombosis triggered by laser-induced intimal injury in the guinea-pig. Caseinoglycopeptide (residues 106-169), the undecapeptide (residues 106-116) and the pentapeptide KNQDK (residues 112-116) from cow kappa-casein, were anti-aggregating peptides and exerted a significant antithrombotic activity in the guinea-pig. Caseinoglycopeptides from three species (cow, ewe and human) were also antithrombotic and the most potent being the human one. The antithrombotic activity was achieved in vivo for doses less than the one suspected from in vitro data and for which, ex vivo platelet aggregation was not decreased. In conclusion, the relative involvement of the fibrinogen gamma-chain C-terminal dodecapeptide could be much more important in in vivo thrombosis process than in in vitro platelet aggregation. Its specificity and activity in vivo unveiled an interesting potential way for inhibition of arterial thrombosis if alternative molecular presentation (i.e. peptidomimetics) and alternative route (i.e. per os) can be developed.

Fibrinogen binding and ADP-induced aggregation in platelets from diabetic subjects

The factors responsible for the platelet hyperaggregability often found in diabetics are obscure. Since fibrinogen is essential for ADP-induced aggregation and specific fibrinogen receptors exist on the platelet membrane, ADP-induced platelet aggregation and fibrinogen binding were measured in 16 diabetics (8 background and 7 proliferative retinopathy, 1 maculopathy) and 9 non-diabetic controls. The velocity of platelet aggregation induced by a range of concentrations of ADP was measured and the 1/Km (1/μM ADP) derived from a Lineweaver-Burk plot. 125I-fibrinogen binding was measured in washed platelets and expressed as per cent total radioactivity. Metabolic control of diabetes was assessed by HbA1. Mean per cent specific fibrinogen binding 15 min after the addition of ADP was significantly increased in the 8 diabetics with severe retinopathy compared to controls (5.08 ± 1.04 SEM vs 3.04 ± 0.53: p<0.05). Platelets from diabetics with and without severe retinopathy were significantly more sensitive to ADP-induced aggregation (mean 1/Km0.74±0.13 SEM vs 0.36 ±0.08; p<0.05) and a highly significant correlation with HbA1 was found (r + 0.79; p<0.001). There was no correlation between percentage bound fibrinogen and 1/Km. Thus the increased fibrinogen binding found in these diabetics appears to be associated with the presence of retinopathy, whereas the increased sensitivity of platelets to ADP may also be related to poor metabolic control.

Inheritance of the human platelet alloantigen, PlA1, in type I Glanzmann's thrombasthenia.

The hereditary of the human platelet alloantigen, PlA1, has been studied in Glanzmanns thrombasthenia. The PlA1 content of platelets from three patients, 20 kindred of these patients, including parents and siblings, and 15 unrelated normal individuals was determined using immunologic techniques based on the release of 51Cr from labeled platelets. The amount of membrane glycoproteins (GP) IIb and IIIa in the platelets of these individuals was determined by quantitative crossed immunoelectrophoresis of Triton X-100 soluble proteins using a multispecific rabbit antibody raised against normal platelets. Platelets from the three thrombasthenic patients contained neither detectable GP IIb and GP IIIa nor detectable PlA1 antigen. Platelets from seven kindred with normal amounts of GP IIb and GP IIIa contained PlA1 antigen levels identical to those detected in platelets of normal individuals. Platelets from 13 kindred, including each parent studied, were shown to contain an amount of GP IIb and GP IIIa equivalent to 53% of that amount detected on normal platelets. Platelets from the same individuals expressed amounts of PlA1 antigen that were either 54.0 +/- 4.1 (mean +/- SD) or 28.0 +/- 2.7% of that present on platelets of normal individuals homozygous for the Al allele. The results presented in this report provide evidence that the expression of the thrombasthenic glycoprotein abnormality and the inheritance of PlA1 antigen are controlled by different genes. These results further suggest that lack of expression of the PlA1 antigen on thrombasthenic platelets results from the decrease or absence of the glycoprotein carrier of the PlA1 determinant, previously shown to be GP IIIa.

In vivo effect of platelet factor 4 (PF4) and tetrapeptide AcSDKP on haemopoiesis of mice treated with 5‐fluorouracil

In vivo effects of platelet factor 4 (PF4) and tetrapeptide N‐acetyl‐Ser‐Asp‐Lys‐Pro (AcSDKP) on haemopoietic progenitors were studied in mice treated with 5‐fluorouracil (5‐FU). The mice were injected with PF4 (40u2003μg/kg) or AcSDKP (4u2003μg/kg) twice at 6u2003h intervals, and 20u2003h after the second injection they were given one injection of 5‐FU (150u2003mg/kg). 6, 8 and 13u2003d later the high proliferative potential‐colony forming cell (HPP‐CFC), burst‐forming unit erythroid (BFU‐E), colony forming unit granulocyte‐macrophage (CFU‐GM) colony forming unit megakaryocyte (CFU‐MK), and megakaryocytes (MK) were examined. The results showed that the administration of PF4 or AcSDKP resulted in a significant increase in the number of HPP‐CFC on days 6–8 and BFU‐E and CFU‐GM on day 8 when compared to 5‐FU alone. Furthermore, PF4 was found to increase significantly the number of CFU‐MK and MK on day 8, which was not observed with AcSDKP. However, both molecules had no obvious effect on peripheral blood cells. These data indicate that PF4 or AcSDKP accelerate the recovery in vivo of HPP‐CFC, CFU‐GM and BFU‐E after 5‐FU treatment but their effect may be different on megakaryocytic progenitors and suggests that both molecules may have a haemoprotective effect against chemotherapeutic agents.

A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard-Soulier syndrome abolishes surface expression of the GPIb-V-IX complex

Summary. This paper describes the molecular defect of the second case of Bernard–Soulier syndrome, initially reported in 1957. Analysis of the patients platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib–V–IX complex and revealed small amounts of intracellular GPIbα, GPIbβ and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard–Soulier syndrome. The change occurred in a prototypic alpha‐helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co‐transfection of GPIXPro7 with normal GPIbα and GPIbβ into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbα and GPIbβ, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb–IX complex.

Gray platelet syndrome. Dissociation between abnormal sorting in megakaryocyte alpha-granules and normal sorting in Weibel-Palade bodies of endothelial cells.

The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which megakaryocytes and platelets are deficient in alpha-granule secretory proteins. Since the Weibel-Palade bodies (WPB) of endothelial cells as well as the alpha-granules contain the von Willebrand Factor (vWF) and P-selectin, we examined by transmission electron microscopy the dermis capillary network of two patients with GPS. Endothelial cells showed the presence of normal WPB with typical internal tubules. Using single and double immunogold labeling for vWF and P-selectin, we detected vWF within WPB, where it was codistributed with the tubules, whereas P-selectin delineated the outline of WPB. Therefore, the fundamental targeting defect in GPS is specific to the megakaryocytic cell line.