Evaluation of a diagnostic platelet aggregation test strategy for platelet rich plasma samples with low platelet counts.

INTRODUCTION: Light transmission aggregometry (LTA) is important for diagnosing platelet function disorders (PFD) and von Willebrand disease (VWD) affecting ristocetin-induced platelet aggregation (RIPA). Nonetheless, data is lacking on the utility of LTA for investigating thrombocytopenic patients and platelet rich plasma samples with low platelet counts (L-PRP). Previously, we developed a strategy for diagnostic LTA assessment of L-PRP that included: (1) acceptance of referrals/samples, regardless of thrombocytopenia severity, (2) tailored agonist selection, based on which are informative for L-PRP with mildly or severely low platelet counts, and (3) interpretation of maximal aggregation (MA) using regression-derived 95% confidence intervals, determined for diluted control L-PRP (C-L-PRP). METHODS: To further evaluate the L-PRP LTA strategy, we evaluated findings for a subsequent patient cohort. RESULTS: Between 2008 and 2021, the L-PRP strategy was applied to 211 samples (11.7% of all LTA samples) from 192 unique patients, whose platelet counts (median [range] × 109 /L) for blood and L-PRP were: 105 [13-282; 89% with thrombocytopenia] and 164 [17-249], respectively. Patient-L-PRP had more abnormal MA findings than simultaneously tested C-L-PRP (p-values <0.001). Among patients with accessible electronic medical records (n = 181), L-PRP LTA uncovered significant aggregation abnormalities in 45 (24.9%), including 18/30 (60%) with <80 × 109 platelets/L L-PRP, and ruled out PFD, and VWD affecting RIPA, in others. The L-PRP LTA strategy helped diagnose VWD affecting RIPA, Bernard Soulier syndrome, familial platelet disorder with myeloid malignancy, suspected ITGA2B/ITGB3-related thrombocytopenia, and acquired PFD. CONCLUSION: Diagnostic LTA with L-PRP, using a strategy that considers thrombocytopenia severity, is feasible and informative.

Update on platelet procoagulant mechanisms in health and in bleeding disorders.

Platelet procoagulant mechanisms are emerging to be complex and important to achieving haemostasis. The mechanisms include the release of procoagulant molecules from platelet storage granules, and strong agonist-induced expression of procoagulant phospholipids on the outer platelet membrane for tenase and prothrombinase assembly. The release of dense granule polyphosphate is important to platelet procoagulant function as it promotes the activation of factors XII, XI and V, inhibits tissue factor pathway inhibitor and fibrinolysis, and strengthens fibrin clots. Platelet procoagulant function also involves the release of partially activated factor V from platelets. Scott syndrome has provided important insights on the mechanisms that regulate procoagulant phospholipids expression on the external platelet membrane, which require strong agonist stimulation that increase cystolic calcium levels, mitochondrial calcium uptake, the loss of flippase function and activation of the transmembrane scramblase protein anoctamin 6. There have been advances in the methods used to directly and indirectly assess platelet procoagulant function in health and disease. Assessments of thrombin generation with platelet rich plasma samples has provided new insights on how platelet procoagulant function is altered in inherited platelet disorders, and how platelets influence the bleeding phenotype of a number of severe coagulation factor deficiencies. Several therapies, including desmopressin and recombinant factor VIIa, improve thrombin generation by platelets. There is growing interest in targeting platelet procoagulant function for therapeutic benefit. This review highlights recent advances in our understanding of platelet-dependent procoagulant mechanisms in health and in bleeding disorders.

Thrombin generation abnormalities in commonly encountered platelet function disorders.

INTRODUCTION: Studies of thrombin generation (TG) with platelet-rich plasma (PRP) and platelet-poor plasma (PPP) have provided insights on bleeding disorders. We studied TG for a cohort with commonly encountered platelet function disorders (PFD). METHODS: Participants included 40 controls and 31 with PFD due to: nonsyndromic dense granule (DG) deficiency (PFD-DGD, n = 9), RUNX1 haploinsufficiency (n = 6) and aggregation defects from other, uncharacterized causes (n = 16). TG was tested with PRP and PPP samples. As DG store ADP and polyphosphate that enhance platelet-dependent TG, PFD-DGD PRP TG was tested for correction with ADP, polyphosphate and combined additives. Tissue factor pathway inhibitor (TFPI), platelet factor V (FV), and platelet TFPI and ANO6 transcript levels were also evaluated. Findings were tested for associations with TG endpoints and bleeding. RESULTS: PFD samples had impaired PRP TG, but also impaired PPP TG, with strong associations between their PRP and PPP TG endpoints (P ≤ .005). PFD-DGD PRP TG endpoints showed associations to PPP TG endpoints but not to DG counts, and were improved, but not fully corrected, by adding polyphosphate and agonists. PFD participants had increased plasma TFPI and reduced platelet TFPI (P ≤ .02) but normal levels of platelet FV, and platelet TFPI and ANO6 transcripts levels. PFD plasma TFPI levels showed significant association to several PPP TG endpoints (P ≤ .04). Several PFD PRP TG endpoints showed significant associations to bleeding symptoms, including wound healing problems and prolonged bleeding from minor cuts (P ≤ .04). CONCLUSION: TG is impaired in commonly encountered PFD, with their PRP TG findings showing interesting associations to symptoms.

Update on diagnostic testing for platelet function disorders: What is practical and useful?

INTRODUCTION: Platelet function disorders (PFD) are an important group of bleeding disorders that require validated and practical laboratory strategies for diagnosis. METHODS: This review summarizes the authors' experiences, current literature, and an international survey to evaluate the practices of diagnostic laboratories that offer tests for PFD. RESULTS: Blood counts, blood film review, and aggregation tests are the most commonly performed investigations for PFD and help determine whether there is thrombocytopenia and/or defective platelet function due to a variety of causes. The performance characteristics of tests for PFD, and the level of evidence that these tests detect bleeding problems, are important issues to determine where tests are useful for diagnostic or correlative purposes, or research only uses. Platelet aggregation assays, and quantitative analysis of platelet dense granule numbers, are tests with good performance characteristics that detect abnormalities associated with increased bleeding in a significant proportion of individuals referred for PFD investigations. Lumiaggregometry estimates of platelet adenosine triphosphate release show greater variability which limits the diagnostic usefulness. Diagnostic laboratories report that fiscal and other constraints, including a lack of high-quality evidence, limit their ability to offer an expanded test menu for PFD. CONCLUSION: PFD are clinically important bleeding disorders that remain challenging for diagnostic laboratories to investigate. While some PFD tests are well validated for diagnostic purposes, gaps in scientific evidence and resource limitations influence diagnostic laboratory decisions on which PFD tests to offer.

Quebec platelet disorder: update on pathogenesis, diagnosis, and treatment.

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with reduced platelet counts and a unique gain-of-function defect in fibrinolysis due to increased expression and storage of urokinase plasminogen activator (uPA) by megakaryocytes. QPD increases risks for bleeding and its key clinical feature is delayed-onset bleeding, following surgery, dental procedures or trauma, which responds only to treatment with fibrinolytic inhibitors. The genetic cause of the disorder is a tandem duplication mutation of the uPA gene, PLAU, which upregulates uPA expression in megakaryocytes by an unknown mechanism. The increased platelet stores of uPA trigger plasmin-mediated degradation of QPD α-granule proteins. The gain-of-function defect in fibrinolysis is thought to be central to the pathogenesis of QPD bleeding as the activation of QPD platelets leads to release of uPA from α-granules and accelerated clot lysis. The purpose of this review is to summarize current knowledge on QPD pathogenesis and the recommended approaches to QPD diagnosis and treatment.

A prospective cohort study of light transmission platelet aggregometry for bleeding disorders: is testing native platelet-rich plasma non-inferior to testing platelet count adjusted samples?

Light transmission platelet aggregometry (LTA) is important to diagnose bleeding disorders. Experts recommend testing LTA with native (N) rather than platelet count adjusted (A) platelet-rich plasma (PRP), although it is unclear if this provides non-inferior, or superior, detection of bleeding disorders. Our goal was to determine if LTA with NPRP is non-inferior to LTA with APRP for bleeding disorder assessments. A prospective cohort of patients, referred for bleeding disorder testing, and healthy controls, were evaluated by LTA using common agonists, NPRP and APRP (adjusted to 250 x 109 platelets/l). Recruitment continued until 40 controls and 40 patients with definite bleeding disorders were tested. Maximal aggregation (MA) data were assessed for the detection of abnormalities from bleeding disorders (all causes combined to limit bias), using sample-type specific reference intervals. Areas under receiver-operator curves (AUROC) were evaluated using pre-defined criteria (area differences: < 0.15 for non-inferiority, > 0 for superiority). Forty-four controls and 209 patients were evaluated. Chart reviews for 169 patients indicated 67 had bleeding disorders, 28 from inherited platelet secretion defects. Mean MA differences between NPRP and APRP were small for most agonists (ranges, controls: -3.3 to 5.8; patients: -3.0 to 13.7). With both samples, reduced MA with two or more agonists was associated with a bleeding disorder. AUROC differences between NPRP and APRP were small and indicated that NPRP were non-inferior to APRP for detecting bleeding disorders by LTA, whereas APRP met superiority criteria. Our study validates using either NPRP or APRP for LTA assessments of bleeding disorders.

An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts.

Light transmission platelet aggregation tests are important for diagnosing platelet function defects. However, uncertainties exist about the best procedures to determine aggregation reference intervals. We investigated methods for determining reference intervals for light transmission aggregation tests, using the % maximal aggregation values for prospectively collected data on healthy control samples. Reference intervals for samples tested at 250 x 10(9) platelets/l were determined by mean +/- 2 standard deviations and non-parametric analyses. To establish reference intervals for tests on thrombocytopenic subjects, regression analyses were used to estimate 95% confidence limits for % maximal aggregation, according to sample platelet counts, using data for control samples diluted to match the platelet count of undiluted thrombocytopenic patient platelet-rich plasma samples. For samples tested at 250 x 10(9) platelets/l, non-parametric analyses described 95% of data for healthy control samples better than mean +/- 2 standard deviations. For samples tested at lower counts, to match thrombocytopenic samples, the % maximal aggregation was influenced by platelet count and derived limits were wider at very low platelet counts for almost all agonists. With ristocetin, it proved feasible to test samples with very low platelet counts to exclude Bernard-Soulier syndrome and type 2B von Willebrand disease. Non-parametric analyses should be the preferred method to establish light transmission aggregation reference intervals for samples tested at normal platelet counts. The derived limits for thrombocytopenic samples provide guidance for evaluating thrombocytopenic platelet function disorders, including which agonists to test, based on the sample platelet count.

Endocytosis and storage of plasma factor V by human megakaryocytes.

Factor V is an essential coagulation cofactor that circulates in plasma and platelet alpha-granules where it is stored complexed to multimerin I (MMRN1). To gain insights into the origin and processing of human platelet factor V, and factor V-MMRN I complexes, we studied factorV in cultured megakaryocytes. Factor V mRNA was detected in all megakaryocyte cultures. However, like albumin, IgG and fibrinogen, factorV protein was detectable only in megakaryocytes cultured with exogenous protein. The amount of factor V associated with megakaryocytes was influenced by the exogenous factorV concentration. Similar to platelet factor V, megakaryocyte factor V was proteolyzed and complexed with megakaryocyte-synthesized MMRN1. With secretagogues, megakaryocytes released factor V, IgG, fibrinogen and MMRN1. Immunofluorescent and electron microscopy confirmed factorV uptake by endocytosis and its trafficking to megakaryocyte alpha-granules. These data provide direct evidence that human megakaryocytes process plasma-derived factor V into alpha-granules and generate factorV-MMRN I complexes from endogenously and exogenously synthesized proteins.

Human platelets contain forms of factor V in disulfide-linkage with multimerin.

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

Prospective screening of 205 patients with ITP, including diagnosis, serological markers, and the relationship between platelet counts, endogenous thrombopoietin, and circulating antithrombopoietin antibodies.

Immune thrombocytopenia purpura (ITP) is characterized by destruction of circulating platelets and the presence of antiplatelet antibodies. Many of the current immunomodulatory therapies act by reducing platelet destruction and usually do not have a lasting effect. This prospective, exploratory study characterized patients with ITP by identifying their demographic and comorbid clinical factors, use of treatments, serologic markers of autoimmunity, and possible relationships between platelet counts, concentrations of endogenous thrombopoietin (eTPO), and the presence of circulating anti-TPO antibodies. Data including medical history and laboratory evaluations were collected at a single patient visit on 205 patients (19 children, 186 adults). Reported histories revealed a 5% rate of thrombotic/ischemic events. Autoimmune markers including direct antiglobulin test and antinuclear antibodies were found more frequently than in the normal population; antiplatelet antibody testing was not done. eTPO concentrations were comparable to concentrations found in healthy volunteers. Our study confirmed that no significant inverse correlation occurred between circulating concentrations of eTPO and platelet counts in patients with ITP (Spearman r = -0.15). Two of the 205 patients tested (1%) had neutralizing activity of recombinant human TPO in a biological assay; however, this activity was confirmed to be anti-TPO antibody in only 1 patient.

Studies of alpha-granule proteins in cultured human megakaryocytes.

alpha-Granule protein storage is important for producing platelets with normal haemostatic function. The low to undetectable levels of several megakaryocyte-synthesized alpha-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. alpha-Granule protein storage in vitro has been studied using other cell types, with differences observed in how some proteins are processed compared to platelets. Human megakaryocytes, cultured from cord blood CD34(+) cells and grown in serum-free media containing thrombopoietin, were investigated to determine if they could be used as a model for studying normal alpha-granule protein processing and storage. ELISA indicated that cultured megakaryocytes contained the alpha-granule proteins multimerin, von Willebrand factor, thrombospondin-1, beta-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant proportion of the alpha-granule protein in megakaryocyte cultures was contained within the cells (averages: 41-71 %), consistent with storage. Detailed analyses of multimerin and von Willebrand factor confirmed that alpha-granule proteins were processed to mature forms and were predominantly located in the alpha-granules of cultured megakaryocytes.Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying alpha-granule protein processing and storage.

Intracellular activation of the fibrinolytic cascade in the Quebec Platelet Disorder.

The 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 alpha-granule proteins. The increased u-PA and proteolyzed plasminogen in QPD platelets led us to investigate possible contributions of intracellular plasmin generation to QPD alpha-granule proteolysis. ELISA indicated there were normal amounts of plasminogen and plasmin-alpha(2)-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 alpha-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 alpha-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 alpha-granule proteins in QPD platelets. The QPD is the only known bleeding disorder associated with chronic, intracellular activation of the fibrinolytic cascade.