Glycocalicin Levels Research Articles

The Association between Platelet Glycocalicin and High Microsatellite Instability in Colorectal Cancer

Background Elevated platelet volume is the risk factor for the development and poor overall survival of colorectal cancer (CRC) patients. Both microsatellite status and platelet glycoprotein Ibα (GPIbα) are related to platelet volume in CRC patients. This study aimed to investigate platelet GPIbα ectodomain (termed glycocalicin) levels among CRC patients and the association between the glycocalicin levels and microsatellite status in CRC. Methods The clinical and laboratory data of 430 CRC patients between January 2018 and December 2018 in Harbin Medical University Cancer Hospital were collected. The microsatellite status was determined with a polymerase chain reaction. The participants were separated into high microsatellite instability (MSI-H) and microsatellite stable (MSS) groups according to microsatellite status. The glycocalicin levels were measured with an enzyme-linked immunosorbent assay, and the cut-off point was determined with the receiver-operating characteristics curve. The clinical and pathological characteristics were collected via electronic medical records. Logistic regression was used to explore the association between glycocalicin and microsatellite status. Results Among the 430 CRC patients enrolled, 64 patients (14.9%) were identified as MSI-H and others as MSS CRC. Glycocalicin levels were significantly reduced in patients with MSI-H than those with MSS. After controlling for potential confounders, logistic regression analysis revealed that glycocalicin levels were independently associated with MSI-H CRC. Conclusions Reduced glycocalicin levels are associated with the MSI-H subtype of CRC. Further research is needed to elucidate the mechanisms of the association between glycocalicin and MSI-H in CRC patients.

Changing profile of platelet activity and turnover indices during treatment response of immune thrombocytopenia.

Both platelet count and function change after treatment of immune thrombocytopenia. Platelet function can be measured by plasma markers, including platelet activity [e.g., soluble P-selectin (sP-selectin) and soluble CD40 ligand (sCD40L)] and platelet turnover markers [e.g., glycocalicin (GC)]. Patients were classified into no response (NR, including new diagnosis), partial response (PR) and complete response (CR). One hundred and sixteen samples (29 CR, 32 PR, 55 NR) from 79 patients were collected. Plasma markers (sP-selectin, sCD40L and GC) were measured by ELISA. Platelet counts and mean platelet volume (MPV) were obtained in the clinical laboratory using GenS System-2. The results showed that responsive patients (PR + CR) had higher levels of sP-selectin (P = 0.026) and sCD40L (P = 0.001). Although there was no difference in MPV (P = 0.077) or GC (P = 0.078), there was a marked decrease of GC index (P < 0.001) in responsive patients. Paired sample analysis showed no difference in sP-selectin, sCD40L, MPV or GC but significant difference in GC index (P = 0.017) between NR and PR. Another paired sample analysis showed no difference in sP-selectin, sCD40L, MPV or GC but significant difference in GC index (P = 0.029) between PR and CR. Patients with refractory and newly diagnosed disease had a significant difference in GC (P = 0.020) and sCD40L (P = 0.001), despite similarly low platelet counts. In conclusion, platelet activity markers (sP-selectin and sCD40L) and GC indices change in parallel with treatment response. Plasma levels of GC and sCD40L may be predictors of treatment response.

Von Willebrand Factor-GP1bα Interactions in Venoarterial Extracorporeal Membrane Oxygenation Patients

Evaluate extracorporeal membrane oxygenation (ECMO) patients' platelet adhesion and aggregation under shear stress and determine whether addition of von Willebrand factor (VWF) concentrate improves platelet function. Also, explore whether reduced platelet adhesion and aggregation is associated with clinical bleeding during ECMO. Prospective observational cohort study with translational component. Academic medical center. Consecutive venoarterial (VA) ECMO patients were screened and 20 patients enrolled. VWF multimers, VWF antigen, ristocetin cofactor activity, and plasma glycocalicin levels were measured and values were compared at study points: ECMO day 1 or 2, day 3, and day 5. Platelet adhesion and aggregation were measured in vitro using the total thrombus analysis system. Platelet function was expressed as area under the flow-pressure curve (AUC). VWF concentrate was added in vitro and the AUC after VWF supplementation (VWF AUC) was compared with baseline AUC. Further, baseline AUCs and VWF AUCs were compared between patients who experienced bleeding during ECMO and those who did not. ECMO patients had high VWF antigen levels, high ristocetin cofactor activity, and large VWF multimer loss. Platelet counts fell over the first 5 days on ECMO, and plasma glycocalicin levels were elevated mildly. ECMO patients had severely low platelet adhesion and aggregation in vitro: median AUC = 5.8 (3.5-9.7) ECMO day 1 or 2, median AUC = 6.3 (5.3-11.1) day 3, and median AUC = 5.5 (4.1-8.1) day 5. There was no significant change in AUC over time (p = 0.47). Addition of VWF concentrate increased the AUC compared to baseline at each point (all p < 0.05), but VWF AUC values remained low. Patients with bleeding during ECMO had a low VWF AUC at all points, whereas those without bleeding had a higher VWF AUC on ECMO day 3. VA ECMO patients have severely impaired platelet function, which improved but did not normalize with VWF concentrate. The data suggest that GP1bα receptor loss of dysfunction also contributes to impaired platelet adhesion and aggregation during ECMO. Based on these findings, clinical bleeding in ECMO patients is unlikely to be correctable with VWF supplementation alone.

Platelet GPIb Ectodomain Shedding – A New Biomarker for GI Bleeding in Continuous Flow VAD Recipients?

Purpose Gastroinstestinal bleeding (GIB) occurs in 30% of continuous flow ventricular assist device (CFVAD) patients. Given that nearly all CFVAD recipients demonstrate a loss of large von Willebrand Factor (vWF) multimers, we sought to examine other factors that can contribute to the risk of GIB events. Methods and Materials Blood samples were obtained on all inpatient days or at outpatient clinic visits from 34 patients receiving Thoratec HeartMate II (25), WorldHeart Levacor (6), or Syncardia temporary Total Artifical Heart (TAH; 3) blood pumping systems. Assays were performed to quantify plasma levels of glycocalicin (the platelet GPIb (vWF receptor) ectodomain), platelet microparticle coagulation activity, thrombin anti-thrombin, Coagulation Factor XII, thromboxane B2. Statistcal analyses were performed using Stastica (Statsoft) to examine assay levels prior to, during, and after confirmed bleeding, thromboembolic (TE), and infectious events, and risk factors based on patient demographics and co-morbidities for adverse events. Results Plasma glycocalin was observed to be elevated compared to pre-operative values in all HeartMate II, TAH, and 3 of the Levacor recipients and in older recipients. A glycocalin value above 4 mg/mL (normal range 0-2 mg/mL) was 92% predictive of a bleeding event. A failure of the glycocalin level to decrease below 2.5 mg/mL or an increase following a bleeding event was 97% predictive of a subsequent bleeding event. There was not a consistent signal which could be determied to predict TE or infectious events with greater than 50% accuracy. Conclusions GIB remains a significant complication of CFVAD support and predicting which patients are at highest risk for GIB events. Glycocalin may be a biomarker to indicate patients at risk for GIB and particularly re-occurent GIB events. The mechanism causing an elevation in platelet GPIb shedding (plasma glycocalicin) deserves further investigation as a potential avenue to elucidate the mechanisms contributing to GIB bleeding risk.

The Use of Immature Platelet Fraction to Assess Thrombopoiesis and Mechanisms of Treatment Effect In Immune Thrombocytopenia (ITP)

AbstractAbstract 2513The pathogenesis of ITP involves accelerated platelet destruction and decreased platelet production. The immature platelet fraction (IPF) from the Sysmex XE2100 autoanalyzer is a marker of platelet reticulocytes, so we used it to explore the mechanisms of effect of treatments of ITP. First, 67 patients with ITP were compared to 100 healthy adult controls to test the Absolute IPF (A-IPF) as a marker of thrombopoiesis. A second study investigated the plasma glycocalicin levels, a soluble external portion of membrane glycoprotein 1bα, in conjunction with A-IPF and IPF% to assess platelet turnover and survival.The mean baseline A-IPF in both ITP study groups (2.8 ×109/L & 8.67 ×109/L) were substantially and significantly lower than the controls (7.80 ×109/L & 10.55 ×109/L) but the IPF% were markedly higher (28.20 ×109/L & 20.83 ×109/L vs 3.20 ×109/L & 4.55 ×109/L. In the first study, A-IPF was further validated clinically by comparing responses to a thrombopoietic agent (TPO-A) and GMA161 (an anti-FcγRIII antibody, inhibiting platelet destruction). A platelet count increment >30 ×109/L or >doubling of baseline and an A-IPF increment >10× 109/L were defined as responses. In the TPO-A sub-group, 19/34 patients had platelet and A-IPF responses; 6/34 patients had platelet responses but no A-IPF response; 8/34 had no platelet or A-IPF responses; 1/34 had an A-IPF response but no platelet response (figure). With GMA161, 2/5 patients had a platelet response without an A-IPF response; 1/5 patients had a response in both platelet count and A-IPF; 2/5 patients had no platelet or A-IPF response. Almost all increases of A-IPF >10×109 and all increases >20×109 were only seen with TPO-A (figure). [Display omitted] These results showed, as expected, that platelet responses to a TPO-A were usually associated with increased A-IPF, whereas those with an inhibitor of FcγR-mediated destruction were usually not. The effects of IV anti-D (Winrho) and IVIG in ITP were studied. 8/8 patients treated with IV anti-D had a platelet response without any A-IPF response. 10/19 patients treated with IVIG had a platelet response without a response in A-IPF, however, 4/19 had platelet and (small) A-IPF responses (figure). These results were consistent when the same patient was studied on different episodes.In a separate study, IPF findings were compared to simultaneous samples of plasma glycocalicin levels (Beer, Blood 1994) in 21 patients. Glycocalicin Index (GCI) is a measure normalized for the platelet count. Mean plasma glycocalicin level was marginally higher in ITP patients than controls: 1.86mcg/ml (0.52–3.91) vs 1.6 mcg/ml (0.79–2.53). However, mean GCI was dramatically higher in the ITP group: 31.36 (0.368–229) vs 1.75 (0.72–2.84). For the separate treatment groups, the mean plasma glycocalicin was greatest in TPO-A treated patients and the mean GCI was greatest in IVIG & prednisolone treated patients. This illustrates the relative importance of effective platelet production and platelet turnover in these therapies. There was a statistically significant (p<0.05) inverse correlation between GCI and platelet count in ITP and there was a positive correlation between plasma glycocalicin and A-IPF, suggesting that the A-IPF was a valid marker of platelet turnover.In summary, the results of A-IPF in ITP patients compared to healthy adult controls and in ITP patients treated with TPO-A and GMA161, demonstrate the utility of this measurement in assessing thrombopoiesis and the mechanism of treatment effect in patients with ITP. Although IVIG and IV anti-D have differing interactions with FcγR, the A-IPF results support the primary effect of anti-D and IVIG to be inhibition of platelet destruction. A small group of patients mounted both platelet and A-IPF increments to IVIG, suggesting that IVIG could also cause an increased effective platelet production. Finally, results with GCI, in a smaller number of patients, further confirmed the validity of the IPF. In the future, A-IPF could be used diagnostically (not explored in this study), for assessing response to treatment, and potentially to select the most effective treatment in individuals. Disclosures:No relevant conflicts of interest to declare.

Platelet production and destruction in liver cirrhosis

Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. Serum TPO was significantly lower in LC (29.9±18.1 pg/ml) compared to controls (82.3±47.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis.

Fluctuations in thrombopoietin, immature platelet fraction, and glycocalicin levels in a patient with cyclic thrombocytopenia

Cyclic thrombocytopenia (CT) is an uncommon disorder characterized by periodic fluctuations in platelet count, resulting in alternating episodes of thrombocytopenia and normal platelet counts. Its pathogenesis remains unclear. Here, we report the case of a 65-year-old man with CT who was initially diagnosed with idiopathic thrombocytopenic purpura (ITP) concomitant with chronic thyroiditis. ITP was diagnosed 12 years ago in the absence of periodic fluctuations in platelet counts. He had been treated with corticosteroids; underwent splenectomy; and subsequently been treated with azathioprine, vincristine, high doses of dexamethasone, and herbal medicine. He had not received antibiotic eradication therapy for Helicobacter pylori because there was no evidence of infection with this organism. Since his symptoms did not improve, the treatments were discontinued, and he was placed under observation. He complained of mild bleeding symptoms, such as petechiae and purpura, which occurred intermittently for several months before he presented to us to undergo detailed investigations. His platelet count varied between 3 9 10/ll and 30 9 10/ll approximately every 3–4 weeks. His white blood cell count and hemoglobin concentration remained unchanged. To elucidate the mechanism underlying this disease, we evaluated the serum thrombopoietin (TPO) level, immature platelet fraction (IPF), plasma glycocalicin index normalized for the individual platelet count (GCI), and T helper (Th)1/Th2 ratio. The serum TPO level was measured with a human TPO ELISA kit (R&D Systems Inc., Minneapolis, MN, USA). The serum levels of TPO, a principal regulator of megakaryogenesis and thrombopoiesis, are thought to be regulated via receptor-mediated clearance by platelets and megakaryocytes. The median serum TPO concentration in healthy individuals is 51 pg/ml. The plasma glycocalicin level was measured with a Glycocalicin EIA kit (Takara Bio Inc., Ohtsu, Japan). The GCI was calculated as proposed by Steinberg et al. [1]: GCI = glycocalicin level 9 250 9 10/individual platelet count (/ll). Glycocalicin is a proteolytic fragment of the a-chain of glycoprotein Ib, a platelet membrane. The GCI reflects the rate of platelet destruction [2]. The mean GCI in healthy individuals is 1.27 (range 0.87–1.77). The IPF was determined to reliably quantify reticulated platelet counts by using a fully automated hematology analyzer XE-2100 equipped with special software IPF Master (Sysmex, Kobe, Japan). Reticulated platelets are RNA-rich immature platelets and are thought to reflect the ability of the bone marrow to produce platelets. Therefore, the IPF, as a surrogate marker of the reticulated platelet count, reflects the rate of thrombopoiesis [3]. The mean IPF in healthy individuals is 2.0% (range 0.5–5.7%). The Th1/Th2 ratio was determined by using flow cytometry analysis to measure the ratio of CD4? cells containing intracellular interferon-c to those containing interleukin-4. The fluctuations in the TPO levels, percentage of IPF, and GCI were synchronous and inversely proportional to those in the platelet counts (Fig. 1). In the thrombocytopenic phase, elevated GCI indicated platelet destruction in this patient. Thrombopoiesis was simultaneously accelerated in response to TPO. Consequently, the IPF, which is as an indicator of thrombopoiesis, increased. In the present case, CT seemed to be closely associated with cyclic increases in platelet destruction. However, the mechanism of cyclic platelet destruction remains to be elucidated. Th1/Th2 balance is T. Yujiri (&) Y. Tanaka M. Tanaka Y. Tanizawa Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi 755-8505, Japan e-mail: yujirit@yamaguchi-u.ac.jp

Morphological and functional platelet abnormalities in Berkeley sickle cell mice

Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37 ± 3.2 vs. 27 ± 1.4, mean ± SD; p < 0.001), in association with moderate thrombocytopenia (505 ± 49 × 10 3/μl vs. 1151 ± 162 × 10 3/μl; p < 0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell–Jolly bodies and “pocked” erythrocytes ( p < 0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5 ± 1% vs. 1 ± 1%; p < 0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease.

Juvenile Cyclic Amegakaryocytic Thrombocytopenia

Cyclic thrombocytopenia is a rare disorder described in adults, characterized by periodic platelet count fluctuations of unknown etiology. The authors describe a boy with cyclic changes in platelet counts ranging from 2 x 10(9)/L to 224 x 10(9)/L with a periodicity of 25 days. Since birth, the patient had periods of bruising. Platelet counts were periodically low during these periods. Thrombopoietin plasma levels oscillated inversely with the platelet count, whereas glycocalicin levels oscillated in phase with the platelets. No oscillation was seen in neutrophil and reticulocyte numbers. The bone marrow showed periodic reduction in megakaryocyte counts. In an in vitro megakaryocytopoiesis assay, the patient's CD34+ cells showed megakaryocyte formation, although to a lower level than controls. Addition of patient plasma, collected during the rise in platelet numbers, to cultures with normal bone marrow-derived CD34+ cells caused an increase in the development of CD41+ megakaryoblasts. Because the periods with bruising had existed since birth, apparently this is a form of congenital cyclic thrombocytopenia. The underlying mechanism of the cyclic thrombocytopenia in this patient is not yet clear, and until now, no therapy has been found for this patient. However, platelet transfusions have resulted in cessation of bleeding during thrombocytopenic periods.

Three parameters, plasma thrombopoietin levels, plasma glycocalicin levels and megakaryocyte culture, distinguish between different causes of congenital thrombocytopenia

Fourteen children with congenital thrombocytopenia were analysed in order to unravel the mechanisms underlying their thrombocytopenia and to evaluate the value of new laboratory tests, namely measurement of plasma thrombopoietin (Tpo) and glycocalicin (GC) levels and analysis of megakaryocytopoiesis in vitro. Three groups of patients were included. The first group (n = 6) was diagnosed with congenital amegakaryocytic thrombocytopenia. They had no megakaryocytes in the bone marrow, three out of four patients showed no megakaryocyte formation in vitro, and all had high Tpo and low GC levels. Mutations in the thrombopoietin receptor gene, c-mpl, were the cause. The second group of patients (n = 3) had normal Tpo and severely decreased GC levels. In bone marrow, normal to increased numbers of atypical, dysmature megakaryocytes were present. In vitro megakaryocyte formation was quantitatively normal. A defect in final megakaryocyte maturation and subsequent (pro-)platelets may be the cause of the thrombocytopenia. The patients in the third group (n = 5) had Wiskott-Aldrich syndrome (WAS). They had normal Tpo and GC levels and normal megakaryocyte formation both in vivo and in vitro. This corresponded with the generally accepted hypothesis that thrombocytopenia in WAS is due to increased platelet turnover. In conclusion, different causes of congenital thrombocytopenia can be distinguished using three parameters: Tpo and GC plasma levels and in vitro analysis of megakaryocytopoiesis. Therefore, these parameters may be helpful in early diagnosis of different forms of congenital thrombocytopenia.

Depression is associated with an increase in the expression of the platelet adhesion receptor glycoprotein Ib

There is a significant association between cardiovascular disease and depression. Previous studies have documented changes in platelets in depression. It is unknown if depression causes functional changes in platelet surface receptors. Therefore, we analyzed (1) the surface expression of glycoprotein (GP)Ib and the integrin receptor α IIbβ IIIa, receptors involved in platelet adhesion and aggregation, (2) CD62 (P-selectin) and CD63, integral granule proteins translocated during platelet activation, (3) platelet aggregation in response to ADP and (4) plasma levels of glycocalicin and von Willebrand factor (vWF), in depressed patients compared to healthy volunteers. Fifteen depressed patients with a Hamilton depression score of at least 22 and fifteen control subjects were studied. Platelets were assessed for surface expression levels of GPIb, α IIbβ IIIa, CD62 and CD63 by flow cytometry. Genomic DNA was isolated to investigate a recently described polymorphism in the 5’ untranslated region of the GPIbα gene. The number of GPIb receptors was significantly increased on the surface of platelets from patients with depression compared to control subjects. Surface expression of CD62 was also significantly increased in the depressed patients versus control subjects. There was no significant difference between depressed patients and healthy volunteers in the surface expression of α IIbβ IIIa or CD63, or in glycocalicin or vWF plasma concentration, or ADP-induced aggregation. There was no difference in allele frequency of the Kozak region polymorphism of the GPIbα gene, which can affect GPIb expression. The results of this study demonstrate that the number of GPIb receptors on platelets are increased in depression and suggest a novel risk factor for thrombosis in patients with depression.

Plasma thrombopoietin levels in patients with chronic renal failure.

Thrombopoietin (Tpo) is the most important regulator of thrombocytopoiesis. The main sites of Tpo production are the liver and the kidney produce Tpo. In the current study, the influence of renal failure on overall Tpo production was evaluated. Tpo levels were measured in 23 patients on hemodialysis (HD) and 16 patients on chronic ambulatory peritoneal dialysis (CAPD). Plasma glycocalicin (GC) levels and platelet counts were measured as parameters of platelet mass and platelet turnover. Platelet counts were significantly lower in the HD group, both before 207+/-98 x 10(9)/l (P<0.001) and after hemodialysis 202+/-102 x 10(9)/l (P<0.001) when compared to healthy controls, 293+/-79 x 10(9)/l. No significant difference was found between platelet counts in patients on CAPD and healthy donors. Mean plasma Tpo levels of HD patients were higher both before 23+/-18 AU/ml (P<0.0001) and after dialysis 25+/-26 AU/ml (P<0.0001), as compared to Tpo levels in healthy controls (11+/-8 AU/ml). Patients on CAPD had significantly higher Tpo concentrations, 29+/-25 AU/ml than healthy controls (P<0.0001). There was no difference in Tpo level between the HD and CAPD group. No correlation was found between Tpo concentration and platelet count, hematocrit, creatinine or uremia levels. The GC concentration was significantly higher in HD patients and CAPD patients when compared to healthy controls. There was no correlation between GC and Tpo level or platelet count. These results confirm the increased platelet turnover in patients with chronic renal failure. Moreover this study shows that the kidney does not seem to play a major role in the overall Tpo production in the body.

Plasma and platelet von Willebrand factor abnormalities in patients with uremia: lack of correlation with uremic bleeding.

Chronic renal failure often is associated with abnormal bleeding that may represent an important complication of this disorder. The hemorrhagic tendency currently is attributed to altered primary hemostasis, mainly platelet dysfunction. However, von Willebrand factor (vWF) also seems to be involved, even though the nature of its abnormalities is still controversial. To gain insight into the role of vWF in determining uremic bleeding, we studied 11 patients with stable, chronic renal failure. We found a significant increase in plasma factor VIII (FVIII), vWF:antigen (Ag), and vWF:ristocetin cofactor (Rco) levels, associated with a mean decrease in platelet vWF:Ag. Plasma vWF multimer pattern was characterized by increased representation of all oligomers in all patients, but five patients also showed a slight decrease in large vWF multimers. In addition, platelet vWF multimer pattern displayed a decrease in all components, especially those with high molecular weight. Despite normal bleeding time, collagen-induced platelet aggregation was defective in almost all patients, whereas vWF collagen binding capacity was normal. The levels of glycocalicin, the circulating fragment of glycoprotein Ib-IX, the major platelet vWF receptor, were also normal. In six patients who also were studied after initiation of dialysis, collagen-induced platelet aggregation was impaired further. Moreover, plasma vWF, and especially FVIII levels, were increased additionally, in association with a normalized platelet vWF content and an improved vWF multimer pattern. The results suggest that vWF abnormalities are present in uremia. Moreover, thrombopathy caused by impaired collagen-induced platelet aggregation is constantly present and apparently not improved by dialytic treatment.

Comparison of glycocalicin, thrombopoietin and reticulated platelet measurement as markers of platelet turnover in HIV+ samples

It is important to distinguish between decreased platelet/megakaryocyte production or increased peripheral platelet destruction as causes of thrombocytopenia. The measurement of reticulated platelets, plasma glycocalicin and thrombopoietin (TPO) levels are potentially of use as discriminators. Thrombocytopenia occurs in many HIV+ patients, and plasma glycocalicin has previously been shown to be elevated in this patient group. Reticulated platelets, glycocalicin and TPO were measured in samples from 56 HIV+ subjects and 20 healthy normal controls. The glycocalicin index (GCI - the glycocalicin levels adjusted for the platelet count) measured in HIV+ subjects was found to be significantly elevated when compared to normal controls (mean GCI 1.5 and 1.27, p = 0.04), while the percentage of reticulated platelets and TPO levels were not. Thrombocytopenic HIV+ subjects had significantly elevated mean GCI (2.8 and 1.4, p < 0.0001), TPO (85.2 and 27.2 pg/ml, p = 0.002), percentage of reticulated platelets (15.3 and 10.8%, p = 0.01), and significantly reduced absolute numbers of reticulated platelets (16.2 and 24.5 2 10 9 /l, p = 0.0004) when compared to non-thrombocytopenic HIV+ subjects. GCI and percentage of reticulated platelets exhibited a significant positive correlation ( r = 0.4, p = 0.002) in HIV+ subjects. The reticulated platelet, TPO and GCI data suggests that thrombocytopenic HIV+ subjects have normal platelet production, and increased peripheral platelet destruction.

Analysis of megakaryocytopoiesis in children with congenital thrombocytopenia

In this study, we analysed 15 children with congenital thrombocytopenia in order to unravel the underlying mechanisms. Plasma thrombopoietin (Tpo) and glycocalicin (GC, a marker for platelet turnover) levels were measured and megakaryocytopoiesis was studied with an in vitro liquid-culture system. Based on clinical and laboratory data included patients were divided into three groups. The first group of seven patients was diagnosed with amegakaryocytic thrombocytopenia. In bone marrow from these patients hardly any megakaryocyte was found and also in in vitro cultures no megakaryocyte formation was observed. Plasma Tpo levels were severely increased and GC levels were decreased, both indicating a platelet production defect. Sequence analysis of the gene encoding the Tpo-receptor, c-mpl, revealed mutations in five of the six patients analysed. Some mutations lead to the formation of a stopcodon and thus to a direct loss of Mpl function. We are currently investigating whether the other mutations, leading to single amino-acid replacements, which do not directly predict loss of Mpl function, are causing disruption of Mpl function or expression. The second group of three patients had normal numbers of atypical megakaryocytes in the bone marrow, showed megakaryocyte formation in vitro, normal Tpo plasma levels and severely decreased GC levels. A defect in megakaryocyte maturation and subsequent proplatelet formation of megakaryocytes is likely the cause of the thrombocytopenia in this group. The third group of five patients was diagnosed with Wiskott Aldrich Syndrome (WAS). They had normal Tpo-, normal GC-levels and megakaryocyte formation both in vivo and in vitro. Thrombocytopenia in this group is due to an increased platelet turnover, that is WAS related. Concluding, with three parameters, Tpo and GC plasma levels and in vitro megakaryocyte culture, a good distinction between different causes of congenital thrombocytopenia in children can be achieved.

Increase in Glycocalicin Levels in Platelet Concentrates Stored in Plasma or Synthetic Medium for 8 Days: Comparison with Other Platelet Activation Markers

Background and Objectives: Glycocalicin (GC) is a proteolytic fragment of GpIb and can conveniently be measured in supernatants of platelet concentrates (PCs) by means of a sandwich ELISA. Because of the convenience of the assay and easy sample storage, we tested its suitability as a sensitive platelet activation parameter during PC storage. Material and Methods: Filtered PCs in plasma or additive solution were made from 5 pooled buffy coats and were subsequently stored during 8 days at 22±2°C. Correlation coefficients (r) were calculated after comparison of GC levels with platelet parameters. Results: A significant increase in GC concentration was found on all subsequent sampling days. PC stored in plasma showed GC levels that correlated well with the soluble P–selectin levels (r = 0.7506), P–selectin (CD62P) expression on platelet membranes (r = 0.8843), morphology scores according to Kunicki (r = –0.7102), lactate concentrations (r = 0.9216), glucose concentrations (r = –0.8913) and β–thromboglobulin (β–TG) concentrations (r = 0.8913). In PCs stored in additive solution, the correlation coefficients with these markers were 0.9209 with soluble P–selectin, 0.7161 with CD62P expression, –0.7474 with morphology score, –0.8908 with glucose concentrations, 0.8923 with lactate concentrations and 0.8908 with β–TG concentrations. Conclusions: The GC concentration correlates well with sensitive platelet (activation) parameters, rendering it a sensitive and convenient parameter for platelet activation.

Increase in Glycocalicin Levels in Platelet Concentrates Stored in Plasma or Synthetic Medium for 8 Days: Comparison with Other Platelet Activation Markers

Abstract Background and Objectives: Glycocalicin (GC) is a proteolytic fragment of Gplb and can conveniently be measured in supernatants of platelet concentrates (PCs) by means of a sandwich ELISA. Because of the convenience of the assay and easy sample storage, we tested its suitability as a sensitive platelet activation parameter during PC storage. Material and Methods: Filtered PCs in plasma or additive solution were made from 5 pooled buffy coats and were subsequently stored during 8 days at 22±2°C. Correlation coefficients (r) were calculated after comparison of GC levels with platelet parameters. Results: A significant increase in GC concentration was found on all subsequent sampling days. PC stored in plasma showed GC levels that correlated well with the soluble P‐selectin levels (r = 0.7506), P‐selectin (CD62P) expression on platelet membranes (r = 0.8843), morphology scores according to Kunicki (r =−0.7102), lactate concentrations (r = 0.9216), glucose concentrations (r =−0.8913) and β‐thromboglobulin (β‐TG) concentrations (r = 0.8913). In PCs stored in additive solution, the correlation coefficients with these markers were 0.9209 with soluble P‐selectin, 0.7161 with CD62P expression, −0.7474 with morphology score, −0.8908 with glucose concentrations, 0.8923 with lactate concentrations and 0.8908 with β‐TG concentrations. Conclusions: The GC concentration correlates well with sensitive platelet (activation) parameters, rendering it a sensitive and convenient parameter for platelet activation.

Clinical and laboratory factors that affect the post-transfusion platelet increment

Transfusion of platelet concentrates (PC) reduced the incidence of fatal hemorrhages in several thrombocytopenic conditions. Unfortunately, long-term platelet supportive care may be complicated by the development of a state of refractoriness, resulting in inadequate recovery of functional platelets. PC handling, clinical conditions of the patients and alloimmunization are the main factors affecting refractoriness. We evaluated the post-transfusion platelet increase in 25 patients (M=6, F=19) with hypomegakaryocytic thrombocytopenia receiving random ABO-compatible PC within 24 h after collection. Quality of PC was assessed by platelet count, pH measuring, LDH release, glycocalicin levels, CD-62 and CD-42b expression. Besides history, clinical status and therapy, we searched for the presence of anti-HLA class 1 and anti-HPA 1-4-5 antibodies. Only six patients (24%) were refractory to PC transfusion, as assessed by a corrected count increment (CCI)<5000. Four of such six patients (67%) had anti-HLA antibodies, as compared to zero of 19 responders ( P<0.02). No other investigated clinical or laboratory feature was significantly different in refractory and responsive patients. Although post-transfusion bleeding time was shorter in responders than in refractory patients (297.33 ± 249.95 versus 673.33 ± 409.96; P<0.02), it did not significantly change even in patients with adequate correct count increment. Our data confirm the importance of anti-HLA antibodies in determining adequate post-transfusion recovery or refractoriness.

Effects of Asymptomatic Abdominal Aortic Aneurysm on the Soluble Coagulation System, Platelet Count and Platelet Activation

Objectives: the aim of the study was to determine the effects of infrarenal asymptomatic abdominal aortic aneurysm (AAA) on platelet count and activation. Design: prospective clinical study in a University Department of Vascular Surgery. Patients: one hundred and five patients with AAA. Thirty-two control patients with symptomatic carotid artery stenoses. Methods: platelet count (PC), plasma glycocalicin levels, prothrombin ratio (PTR), activated partial thromboplastin time (APPT), fibrinogen and D-dimer were measured in 23 patients with AAA and 16 control patients with symptomatic carotid artery stenoses. PC alone was measured in a further 84 patients with AAA and 16 with carotid artery stenoses. Results: PC was below the normal range in 8/105 patients and mean PC (215×109/l,S.D. 47.5) was significantly lower than that of a control population (242×109/l, S.D. 16.8) and patients with carotid disease (269×109/l, S.D. 57). Glycocalicin level was above the normal range in 7/23 patients and the median level (28 fg/plt) was significantly higher than that of a normal population (21.6 fg/plt) and patients with carotid disease (12.3 fg/plt). Fibrinogen levels, PTR and APPT were all within the normal range. One patient had a minimally elevated level of D-dimer. Conclusions: the combination of low PC and high glycocalicin levels suggests that there is increased platelet destruction, most likely due to activation within the aneurysm sac.

Post-DDAVP Thrombocytopenia in Type 2B von Willebrand Disease Is not Associated with Platelet Consumption: Failure to Demonstrate Glycocalicin Increase or Platelet Activation

Thrombocytopenia is frequently reported in type 2B von Willebrand disease (vWD), and thought to be related to the abnormally high affinity of 2B von Willebrand factor (vWF) for platelet GPIb-IX. To gain an insight into the nature of this thrombocytopenia, we measured plasma glycocalicin (GC) levels (as a marker of platelet turnover), and platelet surface expression of the alpha granule protein P-selectin (as a marker of platelet activation) in 9 patients with type 2B vWD before, and in 4 patients also following the infusion of 1-desamino-8-d-arginine vasopressin (DDAVP). Three patients presented a persistent decrease of platelet counts in the resting condition. GC levels were within the normal range, regardless of the platelet counts, in all but one patient who presented, on the other hand, a normal platelet count. Moreover, platelets expressed normal amounts of P-selectin on their surface, regardless of platelet counts. These findings suggest that the thrombocytopenia observed in type 2B vWD is not due to platelet activation and subsequent consumption in circulation. Despite a significant, albeit transient, decrease in platelet count, DDAVP did not induce an increase in plasma GC levels, nor enhance P-selectin expression. These observations indicate that the acute post-DDAVP thrombocytopenia in type 2B vWD is not related to platelet activation and consumption. We advance that the post-DDAVP 2B vWF is hemostatically more active, and able to induce agglutination but not aggregation of circulating platelets. This would explain both the prompt recovery of basal platelet counts after the post-DDAVP decrease, and the lack of reported thrombotic complications in this disorder. Therefore, even though 2B vWF is characterized by an enhanced affinity for the platelet surface, its binding to platelet GPIb-IX in the soluble phase is not able to induce true platelet aggregation: vWF thus appears to be mainly an adhesive protein, rather than an aggregating agent.