Rilzabrutinib, an Oral BTK Inhibitor, in Immune Thrombocytopenia.

BACKGROUND: Rilzabrutinib, an oral, reversible covalent inhibitor of Bruton's tyrosine kinase, may increase platelet counts in patients with immune thrombocytopenia by means of dual mechanisms of action: decreased macrophage (Fcγ receptor)-mediated platelet destruction and reduced production of pathogenic autoantibodies. METHODS: In an international, adaptive, open-label, dose-finding, phase 1-2 clinical trial, we evaluated rilzabrutinib therapy in previously treated patients with immune thrombocytopenia. We used intrapatient dose escalation of oral rilzabrutinib over a period of 24 weeks; the lowest starting dose was 200 mg once daily, with higher starting doses of 400 mg once daily, 300 mg twice daily, and 400 mg twice daily. The primary end points were safety and platelet response (defined as at least two consecutive platelet counts of ≥50×103 per cubic millimeter and an increase from baseline of ≥20×103 per cubic millimeter without the use of rescue medication). RESULTS: Sixty patients were enrolled. At baseline, the median platelet count was 15×103 per cubic millimeter, the median duration of disease was 6.3 years, and patients had received a median of four different immune thrombocytopenia therapies previously. All the treatment-related adverse events were of grade 1 or 2 and transient. There were no treatment-related bleeding or thrombotic events of grade 2 or higher. At a median of 167.5 days (range, 4 to 293) of treatment, 24 of 60 patients (40%) overall and 18 of the 45 patients (40%) who had started rilzabrutinib treatment at the highest dose met the primary end point of platelet response. The median time to the first platelet count of at least 50×103 per cubic millimeter was 11.5 days. Among patients with a primary platelet response, the mean percentage of weeks with a platelet count of at least 50×103 per cubic millimeter was 65%. CONCLUSIONS: Rilzabrutinib was active and associated with only low-level toxic effects at all dose levels. The dose of 400 mg twice daily was identified as the dose for further testing. Overall, rilzabrutinib showed a rapid and durable clinical activity that improved with length of treatment. (Funded by Sanofi; ClinicalTrials.gov number, NCT03395210; EudraCT number, 2017-004012-19.).

Consensus guidelines for the management of adult immune thrombocytopenia in Australia and New Zealand.

INTRODUCTION: The absence of high quality evidence for basic clinical dilemmas in immune thrombocytopenic purpura (ITP) underlines the need for contemporary guidelines relevant to the local treatment context. ITP is diagnosed by exclusions, with a hallmark laboratory finding of isolated thrombocytopenia. MAIN RECOMMENDATIONS: Bleeding, family and medication histories and a review of historical investigations are required to gauge the bleeding risk and possible hereditary syndromes. Beyond the platelet count, the decision to treat is affected by individual bleeding risk, disease stage, side effects of treatment, concomitant medications, and patient preference. Treatment is aimed at achieving a platelet count > 20 × 109 /L, and avoidance of severe bleeding. Steroids are the standard first line treatment, with either 6-week courses of tapering prednisone or repeated courses of high dose dexamethasone providing equivalent efficacy. Intravenous immunoglobulin can be used periprocedurally or as first line therapy in combination with steroids. CHANGES IN MANAGEMENT AS A RESULT OF THIS STATEMENT: There is no consensus on choice of second line treatments. Options with the most robust evidence include splenectomy, rituximab and thrombopoietin receptor agonists. Other therapies include azathioprine, mycophenolate mofetil, dapsone and vinca alkaloids. Given that up to one-third of patients achieve a satisfactory haemostatic response, splenectomy should be delayed for at least 12 months if possible. In life-threatening bleeding, we recommend platelet transfusions to achieve haemostasis, along with intravenous immunoglobulin and high dose steroids.

Characteristics and outcomes of therapy-related myeloid neoplasms after peptide receptor radionuclide/chemoradionuclide therapy (PRRT/PRCRT) for metastatic neuroendocrine neoplasia: a single-institution series.

PURPOSE: Peptide receptor radionuclide/chemoradionuclide therapy (PRRT/PRCRT) is an effective therapy for metastatic neuroendocrine neoplasia (NEN), but therapy-related myeloid neoplasms (t-MN) remain of concern. The study reviewed the clinicopathological features and outcomes of patients who developed t-MN. METHODS: Retrospective analysis of all patients diagnosed with t-MN by 2016 WHO classification, from a cohort of 521 patients who received PRRT/PRCRT over a 12-year period. Molecular next-generation sequencing using an in-house 26-gene panel was performed. RESULTS: Twenty-five of 521 (4.8%) patients were diagnosed with t-MN, including six acute myeloid leukaemia (AML) and 19 myelodysplastic syndrome (MDS). The median time from first cycle PRRT/PRCRT to diagnosis of t-MN was 26 months (range 4-91). Twenty-two of 25 (88%) patients had grade 1-2 pancreatic or small bowel NEN with moderate metastatic liver burden. Six patients (24%) had prior chemotherapy. Median number of PRRT cycles = 5 (22/25 (88%) with concomitant radiosensitising chemotherapy). All 25 patients achieved disease stabilisation (68%) or partial response (32%) on RECIST 1.1 at 3 months post-PRRT. At t-MN diagnosis, all patients presented with thrombocytopenia (median nadir 33 × 109/L, range 3-75) and 17 (68%) remained NEN progression-free. Marrow genetic analysis revealed unfavourable karyotype in 16/25 (66%) patients with tumour protein 53 (TP53) mutation in nine (36%). Azacitidine therapy was utilised in ten eligible patients, while four received induction chemotherapy for AML. The median overall survival from first PRRT was 62 months (19-94), but from t-MN diagnosis was only 13 months (1-56), with death due primarily to haematological disease progression. CONCLUSIONS: The diagnosis of t-MN after PRRT/PRCRT is an infrequent but serious complication with poor overall survival. Most patients present with thrombocytopenia; unfavourable genetic mutations have a poor response to t-MN treatment. Prospective data are needed to explore potential pre-existing genetic factors and predictive biomarkers to minimise the risk of t-MN.

Thrombosis in patients with immune thrombocytopenia: incidence, risk, and clinical outcomes.

Background: There is evidence that patients with immune thrombocytopenia (ITP) are at increased risk of thrombosis. However, the association of clinical- and treatment-related factors with thrombosis remains controversial. Objectives: To evaluate the incidence and impact of risk factors for arterial and venous thromboembolism (VTE) in patients with ITP and characterize the clinical features and management of patients. Methods: We performed a retrospective cohort study (January 1, 2011, to October 30, 2022) of adult patients diagnosed with ITP from an Australian tertiary hospital. The incidence rates of thrombosis were calculated in terms of person-years of follow-up. Multiadjusted Cox regression was used to estimate associations. Results: A total of 220 patients with 1365 person-years of follow-up since ITP diagnosis revealed 26 (11.8%) patients with a total of 37 thrombosis events, 29 (78%) VTE and 8 (22%) arterial thromboembolism (ATE). The incidence rate of thrombosis was 2.71 (95% CI, 1.97-3.72) (0.66 [95% CI, 0.33-1.26] for arterial thromboembolism and 2.05 [95% CI, 1.42-2.95] for VTE) per 100 person-years. Mean age and median time to first thrombosis diagnosis was 56 and 2.13 years, respectively. Age, secondary ITP, lines of therapy, thrombosis risk factors, and thrombopoietin receptor agonist therapy were independently associated with thrombosis. Almost all patients (25 of 26, [96%]) had good ITP disease control prior to thrombosis diagnosis, and antithrombotic therapy was deliverable and well tolerated. Conclusion: Diagnosis of thrombosis in patients with ITP, while infrequent, is of clinical significance. We identified from a heterogeneous real-world cohort that older patients with multiply-treated secondary ITP receiving thrombopoietin receptor agonists are at the highest risk.

PI 3-kinase p110beta: a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.

Tranexamic acid rapidly inhibits fibrinolysis, yet transiently enhances plasmin generation in vivo.

Tranexamic acid (TXA) is a lysine analogue that inhibits plasmin generation and has been used for decades as an antifibrinolytic agent to reduce bleeding. Recent reports have indicated that TXA can paradoxically promote plasmin generation. Blood was obtained from 41 cardiac surgical patients randomly assigned to TXA or placebo before start of surgery (preOP), at the end of surgery (EOS), then again on postoperative day 1 (POD-1) as well as POD-3. Plasma levels of tissue-type plasminogen activator (t-PA), urokinase (u-PA), the plasmin-antiplasmin (PAP) complex, as well as t-PA and u-PA-induced clot lysis assays were then determined. Clot lysis and PAP complex levels were also assessed in healthy volunteers before and at various time points after taking 1 g TXA orally. Surgery induced an increase in circulating t-PA, yet not u-PA at EOS. t-PA levels were unaffected by TXA; however, u-PA levels were significantly reduced in patients on POD-3. t-PA and u-PA-induced clot lysis were both inhibited in plasma from TXA-treated patients. In contrast, PAP complex formation, representing plasmin generation, was unexpectedly enhanced in the plasma of patients administered TXA at the EOS time point. In healthy volunteers, oral TXA effectively blocked fibrinolysis within 30 min and blockade was sustained for 8 h. However, TXA also increased PAP levels in volunteers 4 h after administration. Our findings demonstrate that TXA can actually augment PAP complex formation, consistent with an increase in plasmin generation in vivo despite the fact that it blocks fibrinolysis within 30 min. This may have unanticipated consequences in vivo.

Tranexamic acid modulates the immune response and reduces postsurgical infection rates.

Tranexamic acid (TXA) is an antifibrinolytic agent that blocks plasmin formation. Because plasmin is known to promote inflammatory and immunosuppressive responses, we explored the possibility that plasmin-mediated immunosuppression in patients undergoing cardiac surgery can be directly reversed by TXA and decrease postoperative infection rates. The modulatory effect of TXA on inflammatory cytokine levels and on innate immune cell activation were evaluated with multiplex enzyme-linked immunosorbent assay and flow cytometry, respectively. Postoperative infection rates were determined in patients undergoing cardiac surgery and randomized to TXA (ACTRN12605000557639; http://www.anzca.edu.au). We demonstrate that TXA-mediated plasmin blockade modulates the immune system and reduces surgery-induced immunosuppression in patients following cardiac surgery. TXA enhanced the expression of immune-activating markers while reducing the expression of immunosuppressive markers on multiple myeloid and lymphoid cell populations in peripheral blood. TXA administration significantly reduced postoperative infection rates, despite the fact that patients were being administered prophylactic antibiotics. This effect was independent of the effect of TXA at reducing blood loss. TXA was also shown to exert an immune-modulatory effect in healthy volunteers, further supporting the fibrin-independent effect of TXA on immune function and indicating that baseline plasmin levels contribute to the regulation of the immune system in the absence of any comorbidity or surgical trauma. Finally, the capacity of TXA to reduce infection rates, modulate the innate immune cell profile, and generate an antifibrinolytic effect overall was markedly reduced in patients with diabetes, demonstrating for the first time that the diabetic condition renders patients partially refractory to TXA.

Thrombin overcomes the thrombosis defect associated with platelet GPVI/FcRgamma deficiency.

Fibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRgamma-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRgamma-chain complex (FcRgamma(-/-)). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRgamma is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRgamma was associated with a 30% reduction in thrombus growth. Analysis of FcRgamma(-/-) platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRgamma deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRgamma compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRgamma(-/-) mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRgamma deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.

Importance of temporal flow gradients and integrin alphaIIbbeta3 mechanotransduction for shear activation of platelets.

Disturbances of blood flow play an important role in promoting platelet activation and arterial thrombus formation in stenosed, injured, atherosclerotic arteries. To date, glycoprotein Ib (GPIb) has been considered the primary platelet mechanosensory receptor, responding to increased shear with enhanced adhesive and signaling function. We demonstrate here that von Willebrand factor-GPIb interaction is inefficient at inducing platelet activation even when platelets are exposed to very high wall shear stresses (60 dyn/cm(2)). Rapid platelet activation under flow was only observed under experimental conditions in which transiently adherent platelets were exposed to sudden accelerations in blood flow. Platelet responsiveness to temporal shear gradients was integrin alpha(IIb)beta(3)-dependent and occurred only on a von Willebrand factor substrate, as platelets forming integrin alpha(IIb)beta(3) adhesive contacts with immobilized fibrinogen were unresponsive to sudden increases in shear. The calcium response induced by temporal shear gradients was distinct from previously identified integrin alpha(IIb)beta(3) calcium responses in terms of its transient nature, its requirement for platelet co-stimulation by the P2Y(1) purinergic ADP receptor, and its dependence on the influx of extracellular calcium. Our studies demonstrate a key role for temporal shear gradients in promoting platelet activation. Moreover, they define for the first time the involvement of P2Y receptors in integrin mechanotransduction.

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2.

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.

Signaling role for phospholipase C gamma 2 in platelet glycoprotein Ib alpha calcium flux and cytoskeletal reorganization. Involvement of a pathway distinct from FcR gamma chain and Fc gamma RIIA.

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca2+ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP3) and that treating platelets with the IP3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca2+ flux and platelet shape change. Normal shape change was observed in G alpha q-/- mouse platelets, excluding a role for PLC beta isoforms in this process. However, decreased shape change and Ca2+ mobilization were observed in mice lacking PLC gamma 2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca2+ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca2+ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-Fc gamma RIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcR gamma chain. Taken together, these studies define an important role for PLC gamma 2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.

Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow.

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.