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SummaryPlatelet defects due to hyper-responsive GPIbα causing enhanced VWF interaction, counter-intuitively result in bleeding rather than thrombosis. The historical explanation of platelet/VWF clearance fails to explain mechanisms of impaired haemostasis particularly in light of reported poor platelet binding to fibrinogen. This study aimed to evaluate the defects of platelets with hyper-responsive GPIbα and their contribution to impaired in vivo thrombosis. Using the PT-VWD mouse model, platelets from the hTgG233V were compared to control hTgWT mice. Platelets’ pro-coagulant capacity was evaluated using flowcytometry assessment of P-selectin and annexin V. Whole blood platelet aggregation in response to ADP, collagen and thrombin was tested. Clot kinetics using laser injury thrombosis model and the effect of GPIbα inhibition in vivo using 6B4; a monoclonal antibody, were evaluated. Thrombin-induced platelet P-selectin and PS exposure were significantly reduced in hTgG233V compared to hTgWT and not signifi-cantly different when compared to unstimulated platelets. The hTgG233V platelets aggregated normally in response to collagen, and had a delayed response to ADP and thrombin, when compared to hTgWT platelets. Laser injury showed significant impairment of in vivo thrombus formation in hTgG233V compared to hTgWT mice. There was a significant lag in in vitro clot formation in turbidity assay but no impairment in thrombin generation was observed using thromboelastography. The in vivo inhibition of GPIbα facilitated new – unstable – clot formation but did not improve the lag. We conclude platelets with hyper-responsive GPIbα have complex intrinsic defects beyond the previously described mechanisms. Abnormal signalling through GPIbα and potential therapy using inhibitors require further investigations.Supplementary Material to this article is available online at www.thrombosis-online.com.

SummaryRenal insufficiency is characterized by thrombocytopathy, caused by the accumulation of water soluble and protein bound waste products of protein metabolism, which are not adequately eliminated by the kidney. The kidneys also excrete drugs and their metabolites, which accumulate if dosages are not adjusted to the renal function and may cause clinically relevant bleeding (i. e. synthetic penicillins, vitamin K antagonists, new oral anticoagulants). Therefore, each patients kidney function (GFR) ought to be evaluated by the KDIGO guidelines. The survival of chronic renal patients is lowered by increasing cardiovascular complications. Particularly frequent is non-valvular atrial fibrillation. The recommended prophylaxis with vitamin K antagonists for renal insufficiency is hampered by increased bleeding as well as by augmented (coronary) vascular and valvular calcification. It is not known yet whether prophylaxis with vitamin K may prevent this complication. Conclusion: Because new oral anticoagulants are equally or even more effective and cause less bleeding, they may be favoured in future and even in end-stage renal failure if more is known about dosing, safety and efficacy. The measurement of serum FGF 23 concentration may be helpful as a marker for their use.

SummarySummary: The manifestation of an unclear bleeding tendency in childhood calls for an extended coagulation work-up, particularly when a battered child syndrome is suspected and typical concomitant injuries are absent. The chosen diagnostic tests should be able to detect the presence of relatively common coagulation defects such as von Willebrand syndrome or hemophilia, but also rare diseases such as inherited thrombocytopathies. The PFA-100® test does not help to provide a definite diagnosis especially in cases of mild inherited thrombocytopathies, since in most cases the PFA-100® test results are normal. For this purpose, specific platelet function testing is needed. However, the methods are only available in some coagulation laboratories. Also, other limitations need to be taken into consideration such as pre-analytical problems and difficulties in the interpretation of test results especially in infants.We present two cases that were diagnosed with an aspirin-like defect as an inherited thrombocytopathy, even though their PFA-100 closure times were within the normal range. Based on pathological findings in the platelet aggregometry test, this diagnosis could be made.

Sticky platelet syndrome (SPS) is a thrombophilic thrombocytopathy with familial occurrence and autosomal dominant trait, characterized by an increased in vitro platelet aggregation in response to low concentrations of adenosine diphosphate (ADP) and/or epinephrine (EPI). According to aggregation pattern, three types of the syndrome can be identified (hyperresponse after both reagents, Type I; EPI alone, Type II; ADP alone, Type III). Clinically, the syndrome is associated with both venous and arterial thrombosis. In pregnant women, complications such as fetal growth retardation and fetal loss have been reported. The first thrombotic event usually occurs before 40 years of age and without prominent acquired risk factors. Antiplatelet drugs generally represent adequate treatment. The use of other antithrombotics is usually ineffective and may result in the recurrence of thrombosis. In most patients, low doses of antiplatelet drugs (acetylsalicylic acid, 80-100 mg/d) lead to normalization of hyperaggregability. Combination of SPS with other thrombophilic disorders has been described. Despite several studies investigating platelet glycoproteins' role in platelets' activation and aggregation, the precise defect responsible for the syndrome remains unknown. The aim of this review is to summarize authors' own experience about SPS and the clinical data indexed in selected databases of medical literature (PubMed and Scopus).

Pediatric scurvy is a rare condition characterized by perifollicular petechiae and bruising, hemorrhagic gingivitis and musculoskeletal symptoms, all assumed to be predominantly related to abnormal collagen structure. We report on a 9-year-old autistic boy with vitamin C deficiency due to a highly limited food range presenting with multiple petechiae, gum bleeding and debilitating bone pain, in whom platelet aggregometry revealed a distinctly reduced thrombocyte aggregation, normalizing after vitamin C supplementation. This observation indicates that platelet dysfunction may additionally contribute to the hemorrhagic diathesis in scurvy, and demonstrates that ascorbic acid deficiency should be considered in children with an otherwise unexplained acquired thrombocytopathy.

SummaryThe bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5’ to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient’s platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealedimpaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleedingepisodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect.

SummaryBernard-Soulier syndrome (BSS) is a rare, autosomal recessive inherited bleeding disorder associated with thrombocytopenia, thrombocytopathy and giant platelets. BSS is caused by genetic alterations of the glycoprotein (GP) Ib/V/IX complex.We report on a large Swiss family of whom four family members suffer from BSS. Here, a homozygous missense mutation in position 1829 (A→G) of the GPIX gene constituting a N45S substitution is the cause for the bleeding symptoms. A totalof 38 family members within two generations were analyzed regarding the N45S mutation by DNA sequencing and restriction fragment length polymorphism. The laboratory parameters which are characteristically for BSS such as platelet count, platelet volume and the expression of CD42a (GPIX), CD42b (GPIbα) and CD41 (GPIIb) were measured for all 38 individuals. The four homozygous patients showed bleeding symptoms, thrombocytopenia and giant platelets. In these patients, the expression of CD42a (GPIX), CD42b (GPIbα) was diminished. Interestingly, the intensity of the bleeding symptoms of the 4 homozygous family members seemed to vary although they carry the same mutation. The 24 heterozygous carriers did not differ significantly from their 10 wildtype family members regarding bleeding symptoms and laboratory analysis.

Alpha IIb beta 3 integrin mediates platelet aggregation following its activation. Its absence or dysfunction causes Glanzmann thrombasthenia (GT), an inherited bleeding disorder that is rare worldwide but relatively frequent in several populations with high rates of consanguinity, including Arabs in Israel and Jordan. Cysteine residues in the beta 3 epidermal growth factor (EGF) domains are involved in alpha IIb beta 3 formation and activation. In this study we present a novel Cys549Arg mutation in beta 3 identified in six Jordanian families, which in the homozygous state is manifested by severe GT. The mutation is located in EGF-3 of beta 3 predicting disruption of a conserved disulfide bond between Cys549 and Cys558. Haplotype analysis disclosed a common founder whose age estimate was 120-150 years. Flow cytometry revealed 1-14% of normal alpha IIb beta 3 expression at the patients' platelet surface. The Cys549Arg or artificial Cys549Ser mutations were introduced into a beta 3 expression vector. Co-transfection of baby hamster kidney cells with normal or mutant beta 3 along with normal alpha IIb demonstrated reduced surface expression of alpha IIb beta 3 by both mutants. The mutants were constitutively active as demonstrated by 20-fold increased binding of the ligand-mimetic antibody PAC-1. Immunoblotting and immunoprecipitation experiments showed reduced beta 3 and alpha IIb beta 3 expression and a higher than normal ratio of pro-alpha IIb to mature alpha IIb. Immunofluorescence experiments showed that beta 3 and alpha IIb beta 3 were mostly retained in the endoplasmic reticulum. In conclusion, the novel ancestral mutation found in a cluster of Jordanian GT patients disrupts a conserved Cys549-Cys558 bond which results in reduced production of constitutively active alpha IIb beta 3.

ZusammenfassungRekombinanter Faktor VIIa (rFVIIa) wird über die zugelassenen Indikationen hinaus zunehmend zur Behandlung lebensbedrohlicher Blutungsereignissen bei Versagen der jeweiligen Standardtherapien eingesetzt. Kasuistiken und Fallserien dokumentieren die Wirksamkeit und Sicherheit von rFVIIa zur Blutstillung bei Patienten mit Thrombozytopathien und Thrombozytopenien verschiedener Ursache. Eine Zulassung zur Anwendung von rFVIIa liegt bei Patienten mit Thrombasthenie Glanzmann im Falle eines Refraktärzustandes auf die Transfusion von Thrombozytenkonzentraten vor. rFVIIa-Bolusapplikationen mit Dosierungen zwischen 80 und 120 μg/kg Körpergewicht alle 1,5 bis 3 Stunden werden auch bei Bernard-Soulier-Syndrom, Plättchen- Storage-Pool-Defekten und verschiedenen erworbenen Thrombozytopathien erfolgreich eingesetzt. Bei Thrombasthenie Glanzmann sind zur Gewährleistung einer effektiven Hämostase mindestens drei Bolusgaben notwendig. Bei Thrombozytopenie hat sich bei annähernd der Hälfte der Patienten ein einzelner rFVIIa-Bolus zur Beherrschung sonst unstillbarer Blutungen als ausreichend erwiesen. Dabei kann eine Blutstillung unter rFVIIa auch bei Thrombozytenwerten unter 20 000/μl erreicht werden, wenngleich bei höheren Thrombozytenkonzentrationen eine bessere Wirksamkeit erzielbar ist.

In this study, we report on the characterization of a patient with Glanzmann thrombasthenia (GT). Immunochemical analysis on platelets from the patient showed that the expression of alpha IIb beta 3 was only 25% of that in normal healthy controls, suggesting a case of GT. Functional analysis revealed a total lack of fibrinogen binding capacity. Molecular genetic analysis of the full-length cDNA sequences of alpha IIb and beta 3 subunits showed a novel point mutation C621T in alpha IIb cDNA, leading to a missense substitution of threonine for isoleucine at position 176. Coexpression of normal beta 3 and mutant alpha IIb(1176) isoform in mammalian cells showed a marked reduction in the expression of alpha IIb beta 3 heterodimer when compared to the wild-type and a decreased intracellular level of alpha IIb. The T176 I mutation is located in the N-terminal region in the W3:1-2 connecting strand of the beta-propeller. These data suggest that the N-terminal alpha IIb domain plays an important structural role in the formation of heterodimer and that it is also involved in fibrinogen binding.

SummaryWe describe here a novel mutation in glycoprotein (GP) IX transmembrane region in a patient with Bernard-Soulier syndrome (BSS). Flow cytometric analysis of the patient’s platelets showed that GP Iba and GP IX were expressed at decreased levels. Sequence analysis of the gene coding for GP IX revealed a homozygous (G to A) transition at nucleotide 2113, resulting in a Ala 140 (GCC) to Thr (ACC) replacement in the mature peptide, whereas no defects were found in the coding region of the GP Iba and GP Ib? gene. Allele-specific restriction enzyme analysis using HPYCH4 III revealed that the patient was homozygous and her mother and brother were heterozygous for the defect, and excluded the possibility that the mutation was a polymorphism of GP IX.To clarify the effect of this mutation on the surface expression of the GP Ib/IX complex, we introduced this mutation into the cDNA of GP IX by site-directed mutagenesis and performed in vitro transfection studies with plasmids harboring GP Iba, GP Ib? and wild-type GP IX or mutant GP IX. Mutant GP IX decreased the surface expression of GP Iba and GP IX, whereas both immunostaining and immunoblotting of the transfected Chinese hamster ovary (CHO) cells showed abundant GP Iba and GP IX in the cytoplasm of the CHO cells transfected with plasmids harboring GP Iba, GP Ib? and wild-type GP IX or mutant GP IX These findings indicate that the Ala140→Thr mutation in the transmembrane region of GP IX does not induce intracellular GP Ib/IX complex degradation, but prevents its insertion in the cytoplasmic membrane of platelets and CHO cells.

SummaryThe Quebec Platelet Disorder (QPD) is an unusual bleeding disorder associated with increased platelet stores of urokinase-type plasminogen activator (u-PA) and proteolysis of platelet α–granule proteins. The increased u-PA and proteolyzed plasmino-gen in QPD platelets led us to investigate possible contributions of intracellular plasmin generation to QPD α-granule proteolysis. ELISA indicated there were normal amounts of plasminogen and plasmin-α2-antiplasmin (PAP) complexes in QPD plasmas. Like normal platelets, QPD platelets contained only a small proportion of the blood plasminogen, however, they contained an increased amount of PAP complexes compared to normal platelets (P < 0.005). The quantities of plasminogen stored in platelets were important to induce QPD-like proteolysis of normal α-granule proteins by two chain u-PA (tcu-PA) in vitro. Moreover, adding supplemental plasminogen to QPD, but not to control, platelet lysates, triggered further α-granule protein proteolysis to forms that comigrated with plasmin degraded proteins. These data suggest the generation of increased but limiting amounts of plasmin within platelets is involved in producing the unique phenotypic changes to α-granule proteins in QPD platelets. The QPD is the only known bleeding disorder associated with chronic, intracellular activation of the fibrinolytic cascade.

SummaryWe report the defects responsible for Glanzmann thrombasthenia in two patients showing traces of abnormally migrating platelet β3 in immunoblotting. Using PCR-SSCP and direct sequencing, we identified a novel homozygous mutation in exon 10 of the β3 gene of patient 1 which gave a C457 to Y amino acid substitution. A C542 to R substitution in β3 of patient 2 was previously reported by us. These cysteines are present in EGF-domains 1 and 3 respectively of β3. We therefore constructed mutants carrying substitutions on cysteine residues in each of the first three EGF domains of β3, C457, C495 and C542 respectively. Transient expression of these mutants in COS-7 cells, including the C542 and C547 double mutant, proved that disulfide disruption directly affects cell surface expression of the integrin. We then showed by metabolic (35S) labeling and Endo-H glycosidase treatment that these substitutions strongly affected complex maturation within the cell.

SummaryGlanzmann’s thrombasthenia (GT) is a genetically heterogeneous autosomal recessive syndrome associated with a bleeding tendency. To elucidate molecular basis of GT we have screened for mutations 30 GT patients. On the whole, 21 different candidate causal mutations, 17 in the αIIb and 4 in the β3 gene have been found. Only two (αIIb Pro145Ala and IVS3(−3)-418del) have been previously reported. Nine mutations (42.9%) were likely to produce truncated proteins, whereas the remaining 12 were missense mutations that affected highly conserved residues in αIIb and β3 genes. Six mutations were found in different patients suggesting a possible founder effect. The wide spectrum of expressivity, ranging from mild to severe also among patients carrying the same mutations, provided evidence for a role of different loci or circumstantial factors. In conclusion, we have identified a spectrum of unreported mutations that may be of value to unravel the role of specific regions of αIIb and β3 genes.

New mutations in the β3 integrin subunit have been identified in two unrelated Glanzmann thrombasthenia patients originating from India and Bangladesh. Both patients had histories of excessive bleeding and were found to have Glanzmann thrombasthenia based on absent ADP-induced platelet aggregation. Immunoblotting of platelet lysates of Patient 1 demonstrated reduced levels of αIIb and an unexpected high Mr β3 band of ~260,000, with little or no normal-sized β3. Upon reduction, a weak β3 band of normal Mr was observed. Platelet lysates of Patient 2 demonstrated undetectable levels of β3. Sequence analyses identified homozygous mutations in the β3 genes of both patients. Patient 1 had a C506Y missense mutation resulting in the expression of an unpaired cysteine; we propose that the Mr ~260,000 band is a disulfide-bonded β3 dimer. Patient 2 had an insertion mutation resulting in a frameshift and premature termination. Both mutations affect biogenesis of platelet αIIbβ3 receptors.

SummaryG-proteins play an important role in platelet signal transduction and regulate responses upon activation of G-protein coupled receptors (GPCR). We have previously reported a patient with impaired platelet responses associated with deficiency in platelet Gαq. To understand the molecular basis for this defect, the cDNA sequence encoding Gαq (1080 bp) was obtained by reverse-transcription and polymerase chain reaction of platelet RNA; the cDNA sequence showed no mutations in the patient. Platelet Gαq mRNA levels were decreased by >50% compared to normal subjects; platelet Gαi2 mRNA levels were normal. Neutrophil calcium mobilization and elastase secretion, upon activation with several agonists, and neutrophil Gαq mRNA and protein levels were normal. These studies demonstrate that the patient has a defect in Gαq gene expression in platelets but not neutrophils, possibly due to defects in transcriptional regulation or mRNA stability, and suggest a hematopoietic-lineage specific defect.

SummaryIndividuals with Hermansky-Pudlak Syndrome (HPS) lack platelet dense granules and have no ADP-autocrine response. Despite these platelet deficiencies, HPS patients exhibit a surprisingly mild bleeding phenotype. We hypothesize that activation of the PAR4 thrombin receptor compensates for the lack of an ADP-autocrine response by the P2Y12 ADP receptor in individuals with HPS. Here, we determine that PAR4 activation by thrombin occurs well after ADP release from dense granules in normal individuals. However, the signal from PAR4 stabilizes platelet-platelet aggregate formation in the absence of P2Y12 activation by ADP. Thus, the strong signal emanating from PAR4 during platelet aggregation would provide an explanation for the mild bleeding diathesis of HPS.

ZusammenfassungKrankheiten der primären Hämostase umfassen Thrombozytopenien, Thrombozytopathien und Vaskulopathien. Erwachsenenwerte für Thrombozytenzahlen im peripheren Blut können schon im Fetus beobachtet werden, die Thrombozytenfunktion hingegen scheint altersabhängig zu sein (20). Mit neueren Methoden wie zum Beispiel der Durchflußzytometrie wurde es möglich, die Thrombozytenfunktion besser zu messen. Therapeutische Prinzipien sind abhängig von der Ursache und Art der Thrombozytopenie und/oder der Thrombozytenfunktionsstörung. Die immunthrombozytopenische Purpura (ITP) als Beispiel für einen durch periphere Destruktion verursachten Thrombozytenmangel zeigt im Kindesalter oft einen akuten und im Erwachsenenalter meistens einen chronischen Verlauf. Beide Formen sind gekennzeichnet von vielen ungelösten Problemen der Pathophysiologie, Diagnostik und Therapie, die nur durch große internationale Studien gelöst werden können. Häufig wird ein »kritischer Thrombozytenwert« empirisch definiert und zur Vorbeugung vor schweren Blutungen sowohl bei peripherer Destruktion als auch bei Produktionsstörungen benutzt. Dieser Wert kann jedoch generell nicht empfohlen werden. Die Klinik und diverse Laboranalysen sind zu berücksichtigen, um eine individuelle Indikation für eine Behandlung zu stellen.

When performed with standardized methods and techniques, the bleeding time (BT) depends on variables that physiologically alter primary hemostasis. These variables include number of platelets and platelet function, white and red blood cell counts, vascular factors, hormones, and temperature. Variations within normal limits reflect the in vivo situation and are of no clinical relevance. If the BT is prolonged far above the upper normal limit, however, defects of primary hemostasis have to be anticipated. These include thrombocytopenia or thrombocytopathy, anemia, leukopenia, and deficiencies of plasmatic factors such as von Willebrand factor (vWF), fibrinogen, the lupus anticoagulant, and factor V. The BT can be used as screening test for patients with bleeding symptoms. As a single test, the BT gives the best information in pediatrics, in which defects of primary hemostasis are more common than coagulopathies. In addition, BT can guide the therapy of these patients, because it reflects clinical improvement. When used as a preoperative screening test, BT should be combined with the activated partial thromboplastin time (aPTT) because BT usually does not recognize patients with coagulopathies. With standardized techniques and the knowledge of its merits and limitations, BT is a useful test for diagnosing hemostatic disorders, guiding their therapy, and warning of unexpected bleeding complications during surgery. The BT is especially suited for use in pediatrics for the following reasons: (1) It does not require a venipuncture and is similar to capillary blood sampling if performed with standardized devices adapted for pediatric use; (2) it is an in vivo test informing mostly on defects of primary hemostasis, which are the most common bleeding diatheses in childhood; (3) the results are immediately available; (4) it requires only minimal amounts of blood; and (5) it does not require unphysiological reagents and preparation of the sample. The test requires a highly motivated and experienced operator who knows of the many variables influencing the BT. The interpretation cannot be done without knowledge of the history and physical status of the patient and of the limitations of the BT.

Thrombocytopenia is generally known in its most severe form as acquired immunologic disease. However, in some cases thrombocytopenia is constitutional and may or may not be associated with thrombocytopathy. This review focuses on the clinical and biologic diagnostic criteria of genetic thrombocytopenia, with specific emphasis on the clinical value of the platelet life span and wishes to reiterate the necessity of their identification, as an excessively rapid diagnosis of ITP may be a source of treatment failure. As an example of constitutional thrombocytopenia, we report here 83 familial cases with pure genetic macrothrombocytopenia. They are characterized by the absence of significant bleeding disorders, stable thrombocyte counts higher than 50 x 10(9)/l, platelet macrocytosis, normal platelet function, normal or never increased presence of megakaryocytes, and rarely positive immunological abnormalities. In all cases, platelet life span clearly indicated a defect of production with destruction linked to the ageing population (more than 7 days) and no abnormal sequestration in the spleen or liver. Thus, apart from thrombocytopenia with thrombocytopathy, kinetic studies are necessary in thrombocytopenias whenever clinical parameters do not suggest the presence of excessive platelet destruction.

Rapid diagnosis of the French gypsy mutation in Glanzmann thrombasthenia using high-resolution melting analysis

Essential thrombocythaemia is a chronic myeloproliferative disorder characterized by excessive proliferation of megakaryocytes with sustained increase of platelets count. Thromboembolic complications are major causes of morbidity and mortality. Severe bleeding is less frequent, and is usually seen in patients with very a high platelet count. The cause of bleeding seems to be an acquired platelet dysfunction. Activated recombinant FVII (rFVIIa) has shown to be haemostatically effective in bleeding associated with thrombocytopathy (6–8). Here we report a case, which suggests that rFVIIa might be a therapeutic option for uncontrolled bleeding in patients with essential thrombocythaemia. . . .

SummarySubnormal platelet responses to thromboxane A2 (TXA2) were found in a patient with polycythemia vera, and the mechanism of this dysfunction was analyzed. The patient’s platelets showed defective aggregation and release reaction to arachidonic acid, enzymatically generated TXA2 and synthetic TXA2 mimetics (STA2, U-46619). In contrast, they showed normal responses to thrombin. When the platelet TXA2 receptor was examined with both a 125I-labelled derivative of a TXA2 receptor antagonist ([125I]-PTA-OH) and a 3H-labelled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (Kd) and the maximal concentrations of binding sites (Bmax) of the patient’s platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 failed to induce normal elevation in the. cytoplasmic free calcium ion concentration, phosphatidic acid formation and 40 kD protein phosphorylation in the patient’s platelets, whereas these responses to thrombin were within normal ranges. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) also evoked normal response in the 40 kD protein phosphorylation in the patient’s platelets. These results suggested that the patient’s platelets had TXA2 receptor abnormalities which were characterized by defective transduction of the binding signal to postreceptor reactions after normal TXA2 binding.

SummaryA group of patients with clinical hemorrhagic disease who exhibited a prolonged bleeding time and a short prothrombin consumption time classified as Thrombocytopathy A were studied utilizing procedures involving isolated platelets and purified prothrombin. These frozen and thawed platelet extracts had poor platelet factor 3 activity which was normal after the extracts had been treated with ultrasonic oscillations. The electron microscope studies of the morphology of these platelets were abnormal. It is concluded that these platelets contain adequate amounts of platelet factor 3 but are resistant to disintegration and the activity is only liberated with difficulty.The authors wish to thank Miss Jeanne M. Riddle, M.S. medical technologist in Hematology at Henry Ford Hospital for technical assistance in providing the platelet counts for this investigation.

SummaryStudies on three patients with platelet antibodies associated with thrombocytopenia but with different clinical backgrounds are presented. The first patient had chronic lymphocytic leukemia without known contact with an outside source of platelet antigen. Platelet antibodies were detected before a platelet transfusion was given as a measure of platelet survival. The second patient developed a drug (Diuril) induced thrombocytopathy and thrombocytopenia. The platelets appearing in the circulation immediately following the thrombocytopenia exhibited abnormal qualitative and quantitative ultrastructure and abnormal function of platelet factor 3. This constituents the first drug induced thrombocytopathy observed. The third patient had post-hepatitic cirrhosis with functional and histologic impairment. Possible explanations of antibody production were suggested.The antibodies were demonstrated by the platelet agglutination method of Tullis using normal stored and normal stored irradiated platelets as the source of antigens. Coagulation factors were studied together with the platelet antibodies. Qualitative (platelet thromboplastic function) and quantitative (thrombocytopenia) changes were observed in one patient.Patient’s circulating platelets and agglutinated normal stored and normal stored irradiated platelets were studied under the electron microscope. Morphological changes were observed and described.The sera of the patients and normal controls were centrifuged at 126,000 g for 6 hours and platelet agglutination tests were performed with the top and bottom layers. Increased agglutination in the top layers and decreased agglutination in the bottom layers occurred in the patient’s sera without any changes in the normal sera.