Henry M. Rinder

Pharmacology and Biological Efficacy of a Recombinant, Humanized, Single-Chain Antibody C5 Complement Inhibitor in Patients Undergoing Coronary Artery Bypass Graft Surgery With Cardiopulmonary Bypass

BACKGROUND Cardiopulmonary bypass (CPB) induces a systemic inflammatory response that causes substantial clinical morbidity. Activation of complement during CPB contributes significantly to this inflammatory process. We examined the capability of a novel therapeutic complement inhibitor to prevent pathological complement activation and tissue injury in patients undergoing CPB. METHODS AND RESULTS A humanized, recombinant, single-chain antibody specific for human C5, h5G1.1-scFv, was intravenously administered in 1 of 4 doses ranging from 0.2 to 2.0 mg/kg before CPB. h5G1.1-scFv was found to be safe and well tolerated. Pharmacokinetic analysis revealed a sustained half-life from 7.0 to 14.5 hours. Pharmacodynamic analysis demonstrated significant dose-dependent inhibition of complement hemolytic activity for up to 14 hours at 2 mg/kg. The generation of proinflammatory complement byproducts (sC5b-9) was effectively inhibited in a dose-dependent fashion. Leukocyte activation, as measured by surface expression of CD11b, was reduced (P<0.05) in patients who received 1 and 2 mg/kg. There was a 40% reduction in myocardial injury (creatine kinase-MB release, P=0.05) in patients who received 2 mg/kg. Sequential Mini-Mental State Examinations (MMSE) demonstrated an 80% reduction in new cognitive deficits (P<0.05) in patients treated with 2 mg/kg. Finally, there was a 1-U reduction in postoperative blood loss (P<0. 05) in patients who received 1 or 2 mg/kg. CONCLUSIONS A single-chain antibody specific for human C5 is a safe and effective inhibitor of pathological complement activation in patients undergoing CPB. In addition to significantly reducing sC5b-9 formation and leukocyte CD11b expression, C5 inhibition significantly attenuates postoperative myocardial injury, cognitive deficits, and blood loss. These data suggest that C5 inhibition may represent a novel therapeutic strategy for preventing complement-mediated inflammation and tissue injury.

Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion

Platelets are known to become activated during storage, but it is unclear whether such activation affects recovery or survival after platelet concentrate (PC) transfusion. With the use of flow cytometry to determine the percentage of platelets expressing the alpha‐granule membrane protein 140 (GMP‐140), a known adhesive ligand appearing on the platelet surface after activation, several studies were conducted. These investigations evaluated 1) the occurrence of significant platelet activation over time in PCs (n = 46) stored under standard blood bank conditions; 2) the correlation between platelet activation and platelet recovery in normal subjects after PC storage (n = 12), as assessed by the recovery of Indium‐labeled platelets; and 3) the recovery of activated and unactivated platelets in thrombocytopenic cancer patients transfused with standard PCs (n = 11). It was determined 1) that an increasing duration of storage of PC was associated with increasing platelet activation as measured by the percentage of platelets expressing GMP‐140, progressing from a mean of 4 +/− 2 percent (SD) on the day of collection to a mean of 25 +/− 8 percent by 5 days of storage: 2) that, in normal subjects, posttransfusion recovery of autologous platelets stored for 2 to 4 days and then labeled with In111 was inversely correlated with the percentage of activated platelets in the transfused PC (r = ‐0.55, p = 0.05); and 3) that, when thrombocytopenic patients were transfused with standard PCs, the recovery of the activated platelets in the transfused PCs averaged only 38 +/− 15 percent of the number predicted by the absolute platelet increment. It can be concluded that significant platelet activation occurs with standard platelet storage over 5 days and that activated platelets that express GMP‐140 are preferentially cleared from the circulation after transfusion.

Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice

OBJECTIVE For approximately 5% of autologous transplant recipients and a higher proportion of allogeneic transplant recipients, low level and delayed platelet engraftment is an ongoing problem. Mesenchymal stem cells (MSC), which can be derived from bone marrow as well as other organs, are capable of differentiation into multiple cell types and also support hematopoiesis in vitro. Because cotransplantation of marrow-derived stromal cells has been shown to enhance engraftment of human hematopoietic stem cells, we hypothesized that cotransplantation of MSC could enhance platelet and myeloid cell development. MATERIALS AND METHODS We tested this hypothesis by transplantation of CD34-selected mobilized human peripheral blood stem cells (PBSC) into sublethally irradiated NOD/SCID mice with or without culture-expanded human MSC and evaluated human myeloid, lymphoid, and megakaryocytic engraftment with flow cytometry and in vitro cultures. RESULTS We find that MSC cotransplantation enhances human cell engraftment when a limiting dose (<1 x 10(6)) of CD34 cells is administered. This enhancement is characterized by a shift in the differentiation of human cells from predominantly B lymphocytes to predominantly CD13(+), CD14(+), and CD33(+) myeloid cells with a corresponding increase in myeloid CFU in the marrow. Megakaryocytopoiesis is enhanced by MSC cotransplantation as assessed by an increase in both marrow CFU-MK and circulating human platelets. In contrast, MSC do not affect the percentage of human bone marrow cells that expresses CD34(+). CONCLUSIONS Cotransplantation of human mesenchymal stem cells with CD34(+)-selected hematopoietic stem cells enhances myelopoiesis and megakaryocytopoiesis.

Modulation of Platelet Surface Adhesion Receptors during Cardiopulmonary Bypass

Alterations in platelet receptors critical to adhesion may play a role in the pathogenesis of the qualitative platelet defect associated with cardiopulmonary bypass. Using flow cytometry, we measured changes in the following platelet surface adhesive proteins: the von Willebrand factor receptor, glycoprotein Ib; the fibrinogen receptor, glycoprotein IIb/IIIa; the thrombospondin receptor, glycoprotein IV; the adhesive glycoprotein granule membrane protein 140, whose expression also reflects platelet activation and alpha-granule release; and, as a control, the nonreceptor protein HLA, A,B,C. Glycoprotein Ib decreased during cardiopulmonary bypass (P less than 0.05) and reached a nadir at 72% (P less than 0.05) of its baseline value at 2-4 h after bypass. This decrease correlated (r = 0.76) with the magnitude of platelet activation (alpha-granule release) in any given patient, but even platelets that were not activated demonstrated a decrease in glycoprotein Ib expression. Glycoprotein IIb/IIIa also decreased in both the activated (47% of baseline, P less than 0.01) and unactivated (63% of baseline, P less than 0.01) subsets of platelets at the end of cardiopulmonary bypass. Glycoprotein IV and HLA A,B,C did not decrease, but instead increased 2-4 h after cardiopulmonary bypass (P less than 0.05). We conclude that cardiopulmonary bypass produces selective decreases in surface glycoproteins Ib and IIb/IIIa as well as in platelet activation; that these two alterations are temporally but not necessarily mechanistically linked; and that these changes have the potential to adversely affect platelet function.

Hematologic Profile of Sepsis in Neonates: Neutrophil CD64 as a Diagnostic Marker

OBJECTIVE. The goal was to determine the utility of neutrophil CD64 as a diagnostic marker for sepsis in neonates. METHODS. A prospective study that enrolled consecutive infants with suspected sepsis was performed. Complete blood count with differential, blood culture, and CD64 index measurement were performed, and neutrophil CD64 indices were correlated with the diagnoses of confirmed and suspected sepsis. RESULTS. There were 293 episodes of sepsis evaluations for 163 infants. Infants with sepsis episodes (confirmed or suspected; n = 40) were of greater gestational age (34.7 ± 0.9 weeks), compared with those (n = 123) with no sepsis (32.6 ± 0.5 weeks), but had similar birth weights (2325 ± 200 vs 1969 ± 94 g) and Apgar scores at 1 and 5 minutes. There was no difference in the duration of hospitalization for the 2 groups. As expected, the hematologic profiles of sepsis episodes (n = 128) were characterized by higher white blood cell counts, absolute neutrophil counts, absolute band counts, and immature/total neutrophil ratios but lower platelet counts. Sepsis episodes had higher neutrophil CD64 indices (5.61 ± 0.85 vs 2.63 ± 0.20). For all sepsis episodes, the CD64 index had an area under the curve, in receiver operating characteristic analysis, of 0.74; with a cutoff value of 2.30, the CD64 index in combination with the absolute neutrophil count had the highest negative predictive value (93%) for ruling out sepsis and 95% sensitivity for diagnosing sepsis. For culture-positive sepsis episodes, the CD64 index had the highest area under the curve (0.852) of all hematologic variables, with a sensitivity of 80% and a specificity of 79%, with a cutoff value of 4.02. CONCLUSIONS. Neutrophil CD64 is a highly sensitive marker for neonatal sepsis. Prospective studies incorporating CD64 into a sepsis scoring system are warranted.

Activation in stored platelet concentrates: correlation between membrane expression of P‐selectin, glycoprotein IIb/IIIa, and beta‐ thromboglobulin release

By using two distinct measurements of alpha‐degranulation (surface P‐ selectin [alpha‐granule membrane protein‐140] expression and beta‐ thromboglobulin [beta‐TG] release) and quantitation of glycoprotein (GP) IIb/IIIa surface density, stored platelet concentrates were evaluated to determine a) which method of measuring platelet alpha‐ granule release was more sensitive in detecting early platelet activation; b) whether Day 1 levels of activation predicted the extent of activation or cell lysis on Day 5 of storage; and c) whether changes in surface GPIIb/IIIa density were primarily dependent on platelet activation. By using samples from paired and unpaired units stored for 1, 3, and 5 days, four observations could be made. 1) A flow cytometric assay for the percentage of P‐selectin‐positive platelets was more sensitive for early detection of platelet activation than was measurement of beta‐TG release. This finding was most likely due to enhanced sensitivity in detecting platelets that had undergone partial alpha‐granule release. 2) Total P‐selectin expression correlated with beta‐TG release, which indicated that the extent of alpha‐granule membrane fusion with the external platelet membrane was proportional to the amount of alpha‐granule contents released into the supernatant. 3) All of the activation measurements on Day 1 predicted the activation values, but did not predict the degree of cell lysis (measured by lactate dehydrogenase discharge), on Day 5 of storage. 4) Surface GPIIb/IIIa density was increased on the subset of P‐selectin‐positive platelets as compared with the P‐selectin‐negative subset at all times during storage, but, within each subset, GPIIb/IIIa surface density did not significantly increase over the time of storage.

Platelet alpha-granule release in cocaine users.

BACKGROUND Cocaine use has been associated with arterial occlusion resulting from platelet-rich thrombi and with an accelerated, often atypical atherosclerotic lesion that could be ascribed to platelet activation and platelet alpha-granule release. METHODS AND RESULTS Using a flow cytometric method to quantitate the percent of circulating activated platelets in whole blood (those that express the alpha-granule membrane protein P-selectin), we found that 5 of 25 samples from 12 long-term cocaine users had a baseline level of circulating activated platelets > 3 SD (range, 19% to 60%) above the mean (4.4 +/- 3.7%, mean +/- 1 SD) for 85 nonusers (sample n = 130). This subset resulted in a significantly higher mean baseline level of circulating activated platelets (11.8 +/- 14.4%) for all cocaine users (P = .01). By contrast, cocaine and its metabolites, at concentrations documented as obtainable during in vivo cocaine use (10(-7) to 10(-5) mol/L), had no effect on in vitro platelet activation or aggregation, either directly or in concert with platelet agonists. However, in experiments in which cocaine users received blinded infusions of placebo or cocaine, the mean percent of circulating activated platelets rose significantly (P < .05) after infusion of either placebo (peak 77 +/- 31%) or cocaine (peak 65 +/- 28%), the latter at doses resulting in peak plasma cocaine levels averaging < 10(-6) mol/L. CONCLUSIONS Long-term cocaine use in some subjects is intermittently associated with high basal levels of circulating platelets that have undergone alpha-granule release. The inability of cocaine and its metabolites at concentrations of 10(-7) to 10(-5) mol/L to cause platelet P-selectin expression in vitro in this study, coupled with the acute increase in circulating activated platelets observed in vivo after either cocaine or placebo infusion, suggests that in vivo platelet alpha-granule release associated with cocaine use may occur through indirect rather than direct effects of the drug.

In vitro evaluation of stored platelets: Is there hope for predicting posttransfusion platelet survival and function?

T he ability of transfused platelets to circulate and function is dependent on both the effect of the ex vivo storage lesion that undermines platelet functionality and the status of the in vivo milieu of the transfused individual.1 In hospitalized thrombocytopenic patients, factors affecting platelet consumption may be so strong in their influence on platelet recovery and survival that they may outweigh the effects of in vitro platelet storage.2-4 Nevertheless, it has long been recognized that the ability to predict (pretransfusion) the likely in vivo effectiveness of a specific platelet component would provide a valuable adjunct to current transfusion practice, potentially improving clinical outcome, while simultaneously providing more effective inventory control. Indeed, as new methods of pathogen inactivation move toward possible clinical use5-7 accompanied by the potential for more prolonged, yet still safe, platelet concentrate (PC) storage, the ability to accurately assess clinically meaningful PC quality is once again coming into focus as a clinical need. Stored platelets survive and function after transfusion with varying degrees of success. PCs that have been gently prepared and then immediately transfused without a significant storage interval (within 24-48 hrs of donation) have uniformly high recovery, good survival, and preserved function.8 By contrast, after storage for 7 days, occasional PC units will actually become nonviable, such that these platelets demonstrate virtually no survival after transfusion. Nonviability is most often seen following severe pH and metabolic derangement, in which the platelets lose their ability to maintain normal shape and, as a consequence, lose the evanescent “swirling” pattern and the normal hypotonic shock response seen in functional PCs.9,10 However, the survival and function of most transfused platelets fall somewhere between these two extremes. It has always been an attractive hypothesis that an in vitro assay of platelet function that has demonstrated clinical utility for the diagnosis of congenital and other platelet disorders would also be useful for predicting the potential success of transfusing a particular PC unit. However, although theoretically valuable, it is also true that such a prediction by in vitro assays may be undermined by the fact that stored platelets have the ability to rapidly reverse some aspects of the in vitro platelet storage lesion following in vivo transfusion.11,12 One explanation for this reversibility may be the introduction (by transfusion) of an improved plasma milieu for the previously stored platelets, one that has optimal pH, glucose, and lactate levels.13,14 Hence, in the quest for a predictive assay of in vivo platelet survival and function, it is important to distinguish those measures of stored platelet function that will be rapidly reversible (and thus not clinically relevant) from ones that represent a more permanent change in platelet integrity. Previously described changes in platelets that occur during storage and can contribute to poor platelet function and, by extension, to decreased survival posttransfusion fall into three broadly defined categories: platelet activation; metabolic alterations, both cytosolic and mitochondrial; and platelet senescence. Regarding the latter, normal platelet senescence is most likely a relatively minor component of the storage lesion. In fact, after 5 days of storage, returned platelets can still survive in the host for an additional 5 to 7 days,15 implying that the stored platelet has a total average lifespan greater than it would have had in the normal circulation and further suggesting that factors in the transfusion recipient promote recovery from any senescence-based storage lesion. Measuring the level of stored platelet activation, specifically by CD62P surface expression, initially showed some promise for predicting posttransfusion platelet recovery and survival in humans.16-18 The fact that CD62P represents an important adhesive ligand that can result in binding of the platelet to monocyte-macrophages and other cells provided a rationale for why such a measure might correspond to rapid removal of the platelet from the circulation.19 Unfortunately, however, most clinical studies have demonstrated relatively low correlation coefficients20,21 for stored platelet CD62P expression versus in vivo survival. In addition, animal studies have shown that absence of CD62P in a knockout mouse model does not prolong platelet survival,22 and nonhuman primate platelets have the ability to shed surface CD62P and continue to function in the circulation.23 Hence, membrane neo-expression of CD62P does not condemn the platelet to early removal from the circulation, and measuring surface CD62P expression on stored platelets does not appear to be a clinically useful way to predict subsequent transfusion success. Changes in other platelet surface glycoprotein receptor epitopes, which follow in vitro activation,24 have also failed to show a correlation with CCI TRANSFUSION 2003;43:2-6.

Acquired von Willebrand's disease: a concise review.

Acquired von Willebrands disease (AvWD), an adult‐onset bleeding diathesis, has most commonly been found in patients with an underlying lymphoproliferative disease or monoclonal gammopathy. Other malignancies, autoimmune diseases, hypothyroidism, and drugs have also been associated with AvWD. We have included an illustrative case history of a patient with a bleeding diathesis consistent with AvWD and a monoclonal gammopathy who required emergent cardiac surgery. Our review of the literature determined that most cases of AvWD are due to a circulating antibody that combines with the high molecular weight multimers (HMWM) of von Willebrand factor (vWF). These vWF multimer‐antibody complexes are subsequently cleared from the circulation either by the reticuloendothelial system or by adsorption onto tumor cells. Clearance of the HMWM of vWF thus results in extremely low functional levels and variable antigenic levels. Mixing studies which are traditionally used to diagnose factor inhibitors are useful only if removal of vWF‐antibody complexes can be accomplished in vitro. Treatment with intravenous immunoglobulin has recently been shown to be the most effective therapy for patients with an underlying lymphoproliferative disorder or monoclonal gammopathy. This therapeutic strategy is based on the observed immune complex clearance phenomenon that appears to be operative in most cases. Other AvWD‐associated diseases require treatment specifically directed at the underlying disorder. Am. J. Hematol. 54:139–145, 1997

Atrial fibrillation after cardiac surgery/cardiopulmonary bypass is associated with monocyte activation

Atrial fibrillation (AF) contributes significantly to morbidity and mortality in as many as one-third of patients after cardiac surgery that requires cardiopulmonary bypass (CPB). Recent data suggest that inflammatory infiltration of the myocardium may predispose to AF. We conducted an exploratory pilot study to determine if there was an association between the perioperative leukocyte inflammatory response to cardiac surgery/CPB and postoperative AF. We enrolled 72 patients undergoing cardiac surgery with CPB; all patients were in sinus rhythm before surgery. Leukocyte activation (CD11b upregulation) was perioperatively measured in monocytes and neutrophils (PMN). Preoperative C-reactive protein (CRP) and perioperative neutrophil myeloperoxidase (MPO) were also monitored for inflammation, and troponin I was assayed for perioperative cardiac muscle damage. All markers were evaluated for differences between the subset of patients who developed AF versus those who remained in normal sinus rhythm after surgery. All 72 patients completed the study. Postoperative AF developed in 26 (36%) patients. Perioperative monocyte CD11b upregulation was significantly increased in patients who developed AF (P = 0.01), but increases in PMN CD11b were not significantly associated with AF (P = 0.057). The increase in both monocyte and PMN counts after aortic cross-clamp release was significantly associated with postoperative AF (P = 0.007 and P = 0.005, respectively). By contrast, preoperative CRP and perioperative MPO did not differ between AF and normal rhythm patients. Similarly, the peak value of troponin I did not differ between groups. In this pilot study of cardiac surgery/CPB patients, perioperative upregulation of the monocyte adhesion receptor, CD11b, and higher circulating monocyte and PMN numbers were associated with postoperative AF, suggesting that the induction of cellular inflammation during cardiac surgery/CPB may contribute to this pathophysiology.