ß-1 tubulin R307H SNP alters microtubule dynamics and affects severity of a hereditary thrombocytopenia.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons. OBJECTIVES: We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype ß-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia. PATIENTS/METHODS: Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H ß-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of ß-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP ß-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization. RESULTS: We found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP patients and 76 in SNP patients (both ×10(9)  L(-1) ; P < 0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of ß tubulin; in vitro microtubule recovery assays revealed that cells transfected with R307H SNP ß-1 had significantly impaired microtubule recovery. CONCLUSIONS: Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior.

Inherited thrombocytopenias: the evolving spectrum.

The chapter of inherited thrombocytopenias has expanded greatly over the last decade and many "new" forms deriving from mutations in "new" genes have been identified. Nevertheless, nearly half of patients remain without a definite diagnosis because their illnesses have not yet been described. The diagnostic approach to these diseases can still take advantage of the algorithm proposed by the Italian Platelet Study Group in 2003, although an update is required to include the recently described disorders. So far, transfusions of platelet concentrates have represented the main tool for preventing or treating bleedings, while haematopoietic stem cell transplantation has been reserved for patients with very severe forms. However, recent disclosure that an oral thrombopoietin mimetic is effective in increasing platelet count in patients with MYH9-related thrombocytopenia opened new therapeutic perspectives. This review summarizes the general aspects of inherited thrombocytopenias and describes in more detail MYH9-related diseases (encompassing four thrombocytopenias previously recognized as separate diseases) and the recently described ANKRD26-related thrombocytopenia, which are among the most frequent forms of inherited thrombocytopenia.

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country.

BACKGROUND: Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource-limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina. METHODS: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin-9 in granulocytes and platelet thrombospondin-1 and molecular screening of candidate genes. RESULTS: Thirty-one patients from 14 pedigrees were included; their median age was 32 (4-72) years and platelet count 72 (4-147)×10(9) L(-1). Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9-related disease in four, while classic and monoallelic Bernard-Soulier syndrome, gray platelet syndrome, X-linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. CONCLUSIONS: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.

Platelet size distinguishes between inherited macrothrombocytopenias and immune thrombocytopenia.

BACKGROUND: Distinguishing inherited thrombocytopenias from immune thrombocytopenia (ITP) can be difficult, and patients are therefore at risk of misdiagnosis and inappropriate treatments. Although it is known that the most common inherited forms of thrombocytopenia are characterized by increased platelet size, the diagnostic power of this feature has never been investigated. OBJECTIVES: The aim of this study was to test the hypothesis that platelet size can be used to differentiate ITP from inherited macrothrombocytopenias. PATIENTS/METHODS: We measured mean platelet volume (MPV) and mean platelet diameter (MPD), within 2 h of blood sampling, in 35 patients with inherited macrothrombocytopenias [15 MYH9-related disease (MYH9-RD), three biallelic and 17 monoallelic Bernard-Soulier syndrome (BSS)], and 56 with ITP. Using receiving operating characteristic analysis, we searched for the best cut-off values to differentiate between these conditions. RESULTS: As expected, platelets were larger in inherited macrothrombocytopenias than in ITP. An MPD larger than 3.3 mum differentiated MYH9-RD and BSS from ITP with 0.89 sensitivity and 0.88 specificity, and an MPV larger than 12.4 fL had 0.83 sensitivity and 0.89 specificity. Combining MPD with MPV increased sensitivity and specificity to 0.97 and 0.89, respectively. CONCLUSION: Platelet size evaluation by both an appropriate cell counter and blood film examination is useful for differentiating inherited macrothrombocytopenias from ITP.

Platelet-Independent defect in hemostasis associated with sirolimus use.

Sirolimus is currently used to prevent rejection of solid organ transplant, and sirolimus-eluting stents have shown promise for the prevention of coronary artery restenosis. Thrombocytopenia is a well-known adverse effect of sirolimus limiting its use. Herein we report on a patient in whom sirolimus caused a platelet-independent hemostasis defect. The patient was a 52-year-old woman who underwent renal transplant with consequent normal kidney function. The immunosuppressive regimen included basiliximab, steroids, and cyclosporine induction later shifted to sirolimus and mycophenolate due to biopsy findings of tubular necrosis on day 6 posttransplantation. At discharge the serum creatinine was 0.7 mg/dL. Four months after transplantation the patient was admitted to our hospital because of fever (37.5 degrees C to 38 degrees C), anorexia, and asthenia. Blood analysis showed: creatinine 1.7 mg/dL, Hb 9.6 g/dL, WBC 6 x 10(3)/microL, PLT 123 x 10(3)/microL, liver function tests normal, LDH 720 mU/mL, fibrinogen 628 mg/dL, d-dimer 0.42 ng/mL, FDP > 40 ng/mL, INR 1.10, PT 87%, aPTT 40 seconds. Cultures and tests for infection were negative. Serum sirolimus level was 25.9 ng/mL. The following day the serum creatinine rose to 2.3 mg/dL and diuresis fell to 20 mL/h. Multiple bleeding times (Ivy test) performed before the renal biopsy were repeatedly over 30 minutes (normal 3 to 5 minutes), despite normal platelet count and platelet function studies. There was no spontaneous aggregation and in vitro aggregation was normal (collagen, ADP, adrenalin, and ristocetin induced). Coagulation studies showed a defect in fibrin formation and a reduction of fibrinolysis. Suspension of sirolimus treatment was followed by remission of fever, improvement of renal function (serum creatinine 1.2 mg/dL), and normalization of bleeding time.

Factors influencing post-transfusional platelet increment in pediatric patients given hematopoietic stem cell transplantation.

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) always require platelet transfusions, but the increase in platelet count is often less than expected. Since factors responsible for poor response to platelet transfusions in this clinical setting are largely unknown, we performed a prospective study in 87 consecutive children transplanted in a single institution. The mean 16-h corrected count increment (CCI) of 598 platelet transfusions was 5.76 +/- 8.32 x 10(9)/l. Both before and after HSCT, 13.8% of patients had antibodies against HLA and/or platelet-specific antigens. Univariate analysis identified 12 factors significantly associated with a lower post-transfusion CCI, but only four reached statistical significance in the multivariate analysis. These four factors were concomitant therapy with vancomycin, alloimmunization, use of an Autopheresis cell separator for preparation of platelet concentrates and cytomegalovirus infection. We, therefore, suggest that a better response to platelet transfusions could be obtained by choosing a suitable cell separator, by avoiding the use of vancomycin and by adopting measures that reduce alloimmunization and CMV infection. Moreover, screening patients for HLA and platelet-specific antibodies before HSCT would identify the majority of subjects who will develop alloimmune refractoriness after transplantation and would allow the search for a compatible donor in advance.

Donor-recipient incompatibility at CD31-codon 563 is a major risk factor for acute graft-versus-host disease after allogeneic bone marrow transplantation from a human leucocyte antigen-matched donor.

Disparities at minor histocompatibility antigens (mHA) are thought to be responsible for acute graft-versus-host disease (aGVHD) in patients receiving bone marrow transplantation (BMT) from a human leucocyte antigen (HLA)-matched donor. Although some mHA have been identified in humans, their role in aGVHD has not. Patients (n = 150) receiving a BMT from an HLA-matched donor were investigated for a correlation between aGVHD and donor/recipient incompatibility for seven polymorphisms previously proposed for mHA (HA-1, H-Y, CD31-codon 125, CD31-codon 563, HPA-1, HPA-3 and HPA-5). Only mismatch at CD31-codon 563 predicted grade II-IV aGVHD. The risk derived from CD31-codon 563 mismatch was the same as that derived from the use of bone marrow from an unrelated donor. We suggest that donor/recipient compatibility at CD31-codon 563 should be added to HLA-typing for donor selection and/or adjustment of aGVHD prophylaxis.

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Transfusion of platelet concentrates cryopreserved with ThromboSol plus low-dose dimethylsulphoxide in patients with severe thrombocytopenia: a pilot study.

We have recently reported the possibility of supporting the phase of severe thrombocytopenia after high-dose chemotherapy (HDC) and stem cell transplantation using 5% dimethylsulphoxide (DMSO)-cryopreserved autologous platelet concentrates (PCs). The aim of the present study was to evaluate the therapeutic potential of ThromboSol (a recently developed platelet storage solution) plus PCs cryopreserved in 2% DMSO in patients undergoing myeloablative chemotherapy and autologous transplantation. PCs were collected from 14 women with breast cancer by a single plateletapheresis and cryopreserved in ThromboSol/2% DMSO by either direct insertion in a -80 degrees C freezer or in liquid nitrogen after computer-controlled rate (CR) freezing. When required, PCs were thawed, centrifuged to remove the cryoprotectants and transfused. In vitro studies on thawed platelets showed loss of epitopes of surface glycoproteins and a marked reduction of functional activity compared with fresh platelets. Transfusion of CR-frozen PCs was associated with a mean 1 h corrected count increment (CCI) of 9.2 +/- 5.4 x 109/l and only one allogeneic PC was required in this group. In contrast, six out of seven patients required additional allogeneic transfusions in the -80 degrees C group (CCI = 2.7 +/- 1.4 x 109/l). ThromboSol-treated PCs have the ability to overcome thrombocytopenia if processed by a CR freezing protocol, but appear ineffective when frozen by direct placing at -80 degrees C.

In vitro and in vivo effects of desmopressin on platelet function.

BACKGROUND AND OBJECTIVE: Desmopressin (DDAVP) may shorten bleeding time in patients with disorders of platelet function, but its mechanism of action in these conditions is still a matter of debate. In particular, contrasting results have been obtained concerning the ability of DDAVP to interact with platelets and to activate them directly. To gain further information on the DDAVP-platelet interaction, we studied the in vitro and ex vivo effects of DDAVP on platelet function. DESIGN AND METHODS: Platelet responses to DDAVP both as a single agent and in conjunction with agonists of platelet activation were investigated. For in vitro experiments platelets were obtained from healthy adult volunteers, while the ex vivo effects of DDAVP were studied in 12 patients with a bleeding disorder receiving a test dose of this drug. RESULTS: DDAVP in vitro did not induce either platelet aggregation or surface expression of the activation-dependent antigens; it did, however, greatly inhibit platelet aggregation response to vasopressin (AVP) and increased the maximal extent of platelet aggregation induced by collagen and ADP. DDAVP infusion did not promote the expression of activation antigens, but significantly enhanced ex vivo platelet aggregation stimulated by ADP and collagen. This priming effect was observed in patients with von Willebrand's disease, hemophilia A, May-Hegglin anomaly, gray platelet syndrome and Ehlers-Danlos syndrome. In all these patients bleeding time was shortened by DDAVP infusion. In contrast, neither platelet aggregation nor bleeding time was modified in two subjects with Glanzmann's thrombasthenia. INTERPRETATION AND CONCLUSIONS: Our in vitro experiments indicate that DDAVP interacts directly with platelets and facilitates their activation via other agonists. In vivo results suggest that this effect occurs and is clinically relevant in patients with platelet dysfunction responding to DDAVP with a shortening of bleeding time.

Incompatibility for CD31 and human platelet antigens and acute graft-versus-host disease after bone marrow transplantation.

Bone marrow transplantation (BMT) is often complicated by acute graft-versus-host disease (aGVHD). In patients transplanted with an HLA-matched donor the occurrence of this complication is believed to be favoured by disparities at the minor histocompatibility antigens (mHA). However, few of these polymorphic molecules have been identified. We sought to determine whether donor/recipient incompatibility for HPA-1, HPA-2, HPA-3, HPA-5 or CD31 (codon 125) antigens represented a risk factor for aGVHD and genotyped these antigens in 70 bone marrow donors and their HLA-identical recipients. All patients were children who received BMT for haematological malignancies at a single institution according to well-defined therapy protocols. Statistical analysis showed that incompatibility for CD31 (codon 125) was a risk factor for grade II-IV aGVHD in the overall patient population, whereas HPA-3 incompatibility predicted aGVHD occurrence in HLA-A2 patients only. The magnitude of the aGVHD risk was directly related to the number of HPA/CD31 incompatibilities. No correlation was found between non-identity for HPA/CD31 and aGVHD. Since incompatibility but not non-identity for CD31 or HPA-3 was a risk factor for aGVHD, we suggest that allelic variants of these molecules can serve as mHA in BMT recipients from HLA-identical donors.

Relationship between size and thiazole orange fluorescence of platelets in patients undergoing high-dose chemotherapy.

The reticulated platelet count relies upon the assumption that newly formed platelets contain a residual amount of RNA which selectively binds the dye thiazole orange (TO) and greatly enhances its fluorescence signal. It has, however, recently been shown that almost half of the platelet TO-signal is derived from the labelling of dense-granule nucleotides. It is therefore possible that the higher TO fluorescence of young platelets partially derives from the higher granule content due to their larger volume. To investigate the relationship between platelet size and TO fluorescence we studied 13 patients with high-risk breast cancer undergoing high-dose chemotherapy. Mean platelet volume, platelet distribution width, platelet-large cell ratio, membrane content of glycoprotein Ib and IIb-IIIa and platelet aggregation were significantly greater during resolution than during development of thrombocytopenia, suggesting a prevalence of young and old platelets respectively. Mean TO fluorescence per cell was higher in the platelet population enriched in young cells than in that enriched in old cells, but this difference was no longer observed when the ratio TO signal/platelet size was examined. Moreover, RNase treatment and platelet degranulation reduced TO fluorescence to a similar extent in platelet populations enriched in young or old cells. Therefore our data suggest that the higher TO signal of young platelets is derived, to a significant extent, from their larger volume and granule content.

Autologous platelet collection and storage to support thrombocytopenia in patients undergoing high-dose chemotherapy and circulating progenitor cell transplantation for high-risk breast cancer.

OBJECTIVES: The use of circulating progenitor cell support following high-dose chemotherapy for malignancies decreases but does not entirely abolish platelet transfusion requirement. We investigated the feasibility of supporting the posttransplant thrombocytopenic phase exclusively with autologous platelets collected by apheresis and cryopreserved. METHODS: 25 patients underwent plateletpheresis during the platelet rebound occurring after high-dose cyclophosphamide. Autologous platelets were cryopreserved in 5% dimethylsulfoxide, thawed and transfused during the aplastic phase after the myeloablative regimen whenever clinically required. RESULTS: A single plateletpheresis was carried out in all patients, allowing the harvest of a platelet concentrate with a mean value of 7.7 x 10(11) platelets. No significant procedure- or transfusion-related side effects were recorded. Mean platelet recovery after freezing and thawing was 63% and the mean number of platelet reinfused was 4.8 x 10(11); 23 of 25 patients were fully supported with autologous platelets. CONCLUSION: Plateletpheresis performed in our selected group of patients was found to be a safe and effective procedure to collect large amounts of autologous platelets; the numbers obtained proved to be sufficient for the transfusion demand of almost all patients.

Autologous platelet transfusion in patients receiving high-dose chemotherapy and circulating progenitor cell transplantation for stage II/III breast cancer.

BACKGROUND AND OBJECTIVE: Concerns about the risk of transfusion therapy are driving towards new strategies which are designed to minimize exposure to allogeneic blood products. We aimed to find out whether it is possible to support the phase of thrombocytopenia following high-dose chemotherapy (HDC) and circulating progenitor cells (CPC) transplantation by autologous platelet concentrates (PC). DESIGN AND METHODS: PC were collected from 32 patients undergoing HDC and CPC transplantation for stage II/III breast cancer. A single plateletpheresis was performed at rebound after high-dose cyclophosphamide, when platelet count exceeded 250 x 10(9)/L. PC were cryopreserved in 5% DMSO after controlled-rate freezing and stored in liquid nitrogen. In vitro studies of cryopreserved platelets (aggregation, ATP release and change of mean platelet volume induced by EDTA) were performed. When platelet counts dropped below 20 x 10(9)/L following HDC (thiotepa 600 mg/m2, L-PAM 160 mg/m2) and CPC transplant (CD34+ cells > 5 x 10(6)/kg), PC were thawed in a 37 degrees C water bath, centrifuged to remove DMSO, resuspended in autologous plasma and reinfused within one hour. RESULTS: Large quantities of platelets were harvested in all patients (median 6.6 x 10(11), range 4.8-12.2). In vitro studies showed preserved platelet function as compared to both fresh platelets and standard PC. Twenty-eight out of 32 patients received autologous PC. At the time of transfusion most of the patients were febrile (> 38 degrees C) and had mucositis > G2. The median number of platelets reinfused was 3.8 x 10(11) (range 2.0-8.1) with a median loss during the freeze-thaw-wash procedure of 37%. Autotransfusion was able to maintain platelet count above 20 x 10(9)/L in most patients, with a corrected count increment > 7.5 in 20 cases. Four patients required one additional allogeneic transfusion, two because of a poor increment and two due to a late-occurring epistaxis. No side effects related to PC infusion were recorded. Sixteen control patients who received the same HDC and a similar number of CD34+ cells required a total of 17 allogeneic PC units (1 patient did not require platelet transfusion). INTERPRETATION AND CONCLUSIONS: Our data demonstrate that large doses of autologous platelets can easily be collected and safely administered to support the period of thrombocytopenia in patients undergoing HDC and CPC transplantation. Autologous PC in these patients can abrogate the risks deriving from allogeneic platelet transfusion.

Thrombopoietin levels in patients with primary and reactive thrombocytosis.

Human c-mpl ligand or thrombopoietin (TPO) has been proved to be a critical cytokine in the physiological regulation of thrombopoiesis. Previous evidence suggested that TPO production is constitutive and TPO plasma levels are regulated by the platelet-megakaryocyte mass through c-mpl receptor-mediated uptake and metabolism. To evaluate whether this mechanism of TPO level regulation is also operative in subjects with an elevated platelet count, we evaluated serum TPO in 32 patients with thrombocytosis due to essential thrombocythaemia (ET) or polycythaemia vera (PV) and in 70 subjects with reactive thrombocytosis; 32 healthy subjects were also studied. TPO levels were significantly higher in the ET and PV groups (median 246.2 pg/ml, range 93.5-4596) and reactive thrombocytosis patients (median 287 pg/ml, range 82.7-1960.0) than in normal subjects (median 156.7 pg/ml, range 62.2-352.7). No significant difference was found between the two groups of patients, indicating that serum TPO levels cannot differentiate between primary and reactive thrombocytosis. No significant correlation was found between platelet count and TPO levels in either ET-PV or reactive thrombocytosis, whereas a trend toward a correlation between acute-phase reactants and TPO was observed in patients with reactive thrombocytosis. These results indicate that TPO clearance by platelets-megakaryocytes is not fully operative or is not the only mechanism of TPO level regulation in primitive and reactive thrombocytosis.

Activation of the hemostatic process in patients with unruptured aortic aneurysm before and in the first week after surgical repair.

BACKGROUND AND OBJECTIVE: It has been previously suggested that activation of coagulation and fibrinolysis may sometime occur in patients with unruptured aortic aneurysm. However, the incidence of this complication and the effect of surgical repair are unknown. The objective of our study was to gain further information on this topic. METHODS: We investigated activation of the hemostatic process in 20 consecutive patients with unruptured abdominal aortic aneurysm. We then evaluated the effect of surgical repair of the vascular abnormalities. RESULTS: Both before and in the first week after surgery, the large majority of patients showed clear signs of activation of coagulation (increased plasma levels of prothrombin fragment 1 + 2 and fibrin peptide A), and many had low levels of the natural anticoagulant antithrombin III. Platelets were activated in all cases (high levels of plasma beta-thromboglobulin), and signs of platelet consumption (thrombocytopenia and/or increased mean platelet volume) were present in most of them. INTERPRETATION AND CONCLUSIONS: Activation of the hemostatic process occurs in nearly all patients with abdominal aortic aneurysm and could play a role in the hemorrhagic and thrombotic events that can complicate the clinical development of these subjects.

Platelet composition and function in patients undergoing cardiopulmonary bypass for heart surgery.

BACKGROUND: Previous studies showed severe biochemical and functional damage to platelets in patients undergoing cardiopulmonary bypass for cardiac surgery, and suggested that this derived from the proteolytic action of plasmin on the platelet surface. METHODS: A double-blind study was carried out to compare platelet function and composition in patients randomized to receive the protease inhibitor aprotinin or placebo during reoperation for valvular prosthesis replacement or coronary artery bypass grafting. RESULTS: Flow cytometry with specific monoclonal antibodies and polyacrylamide gel electrophoresis did not show any significant proteolysis of platelet glycoprotein Ib and IIb-IIIa either in the placebo or the aprotinin group. Functional studies were consistent with these results, since ristocetin-induced platelet agglutination was unchanged and platelet aggregation and ATP release induced by collagen and ADP were only slightly reduced by cardiopulmonary bypass. These mild defects in platelet function were partially prevented by aprotinin infusion. CONCLUSIONS: On the basis of our data and those from literature, we suggest that platelets may be affected very little or severely damaged during cardiopulmonary bypass for cardiac surgery, probably depending on some aspects of the technical procedure which remain to be identified. Aprotinin infusion significantly protects platelets in the latter condition, while its role is obviously slight in the former.

Cys209 Ser mutation in the platelet membrane glycoprotein Ib alpha gene is associated with Bernard-Soulier syndrome.

Molecular genetic analysis has been performed on a patient with Bernard-Soulier syndrome (BSS). The patient had characteristically giant platelets and was deficient in the glycoprotein (GP) Ib/IX/V complex, the von Willebrand factor (vWf) receptor on platelets. Previous studies with monoclonal antibodies directed against GP Ib alpha (CD 42b) and GP IX (CD 42a) demonstrated the absence of GP Ib alpha and presence of small amounts of GP IX on the surface of the patient's platelets. In this study the presence of GP V (CD 42d) is also demonstrated. This indicates a defect in the alpha-subunit of glycoprotein Ib. Therefore polymerase chain reaction (PCR)-amplification of the genomic DNA coding for GP Ib alpha was performed. Nucleotide sequence analysis of the entire coding region of GP Ib alpha revealed a homozygous single base pair mutation T-->A, leading to a single amino acid substitution cysteine-->serine at position 209 of the mature protein. We took advantage of the Mse I target site in the mutant allele, created by the T-->A mutation, to analyse all available family members. PCR-ASRA (allele-specific restriction enzyme analysis) using the restriction enzyme Mse I, revealed the heterozygosity of the mother and the two children of the patient, whereas homozygosity of the patient for the Cys209Ser mutation was confirmed. The sister of the patient was not found to be a carrier of the mutant allele. The mutation identified in the family studied, responsible for the deficiency of the GP Ib/IX/V complex, suggests that the cysteine at amino acid position 209 may be involved in disulphide bonding.