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Platelet dysfunction in chemotherapy
acute lymphocytic leukemia (AML/ALL, n=37), multiple myeloma (n=21), malignant lymphoma (n=15) or other hematologic malignancies (n=4). All patients experienced severe thrombocytopenia due to chemotherapy, which was stopped at a median of eight days before blood sample analysis (Table 1). The median age of the patient group was 60 years, and 41% was female (Table 1). Leukocyte and platelet counts were below normal, as was the hemo- globin level. Standard coagulation parameters were deter- mined in plasmas from 43 patients following their chemotherapy treatment. For 70% of the patients, values of activated partial thromboplastin time (aPTT), prothrom- bin time and thrombin time were within reference ranges (Online Supplementary Table S1). Fibrinogen and von Willebrand factor (VWF) levels were slightly elevated, while D-dimer levels were substantially increased in patient plasmas. On the other hand, factor VII activity lev- els were decreased.
Treatment regimens in accordance with national guide- lines varied with disease type.16-18 Since these regimens consisted of multiple chemotherapeutic compounds, the distribution of the drugs was evaluated among patients with different diagnoses. Therefore, the various drugs were assigned to one of five pharmacological classes: A, antitumor antibiotics & topoisomerase II inhibitors; B, antimetabolites; C, alkylating agents; D, mitotic inhibitors; E, other (Online Supplementary Table S2).19 Most patients were treated with anti-tumor antibiotics/topo-isomerase inhibitors, antimetabolites and/or alkylating agents (Online Suplementary Table S3). The patients diagnosed with AML/ALL and lymphoma usually received drugs from one or more of these three classes, while the patients diagnosed with multiple myeloma only received alkylating agents. Of all 77 patients, 50 had undergone hematopoietic stem cell transplantation before inclusion, of which 39 patients received an autologous transplant and 11 an allogenic transplant (Table 1). Blood samples were obtained at eight days (median) after the last administration of chemothera- py or at eight days (median) after stem cell transplantation.
Responsiveness of washed platelets was determined by flow cytometry, using a platelet count of 10x109/L, for 52 patients and 27 healthy control subjects. In the absence of agonists, surface activation markers were low for both patient and control platelets. After stimulation with adeno- sine diphosphate ([ADP]; P2Y1/12 agonist), collagen-relat- ed peptide (CRP-XL; Glycoprotein VI (GPVI) agonist) or thrombin (PAR1/4 agonist) at maximal doses, integrin aIIbβ3 activation (Figure 1A) and P-selectin expression (Figure 1B) of the patients' platelets were reduced to a vari- able extent, when compared to the controls, irrespective of the agonist used.
Detailed analysis indicated that the overall platelet responsiveness (median=36.8% interquartile range [IQR]=29.7- 46.7%), defined as the average fraction of platelets positive for integrin activation and P-selectin expression for the three agonists: (i) was not different between diagnoses, i.e., AML/ALL, multiple myeloma, lymphoma and other hematological malignancies (Kruskal Wallis H test, P=0.192); (ii) was not affected by stem cell transplantation, i.e., no transplant, autologous or allogenic stem cell transplantation (Kruskal Wallis H test, P=0.640); (iii) was similar for the four major treatment classes, i.e., A+B, A+B+C, B+C, C (Kruskal Wallis H test, P=0.512; Online Supplementary Figure S1); and (iv) did not correlate with the whole blood platelet count (Spearman’s
Table 1. Characteristics and hematological parameters of patients dur- ing myelosuppression.
Patients characteristics
Age (years) Female/male (n) Diagnosis (n)
AML/ALL Multiple myeloma Lymphoma
Other
Stem cell transplantation (n) Autologous
Allogeneic
Time since chemotherapy (days)
Time since stem cell transplantation (days)
Number / Value
60 (60) 32/45 (20/32)
37 (25) 21 (12) 15 (13) 4 (2)
39 (26) 11 (8) 8 (9) 8 (8)
Reference range
3.5 - 11.0
7.5 - 11.0 150 - 400
1.1 -6.147
Blood parameters
Leukocyte count (x 109/L)
Hemoglobin (mM)
Platelet count (x 109/L)
Absolute immature platelet
number (x 109/L)
Immature platelet fraction (%)
Value
0.15 (0.22)
5.7 (5.7) 8 (7) 0.31 (0.26)
3.9 (3.6)
Data are for total number of patients (n=77). Patient information for study A (n=52) is indicated between brackets. Median values are given. AML/ALL: acute myeloid leukemia or acute lymphocytic leukemia.
rho=0.175, P=0.239). Together, this suggested that the vari- ability in platelet responsiveness among patients was not directly linked to the disorder, treatment type or number of (residual) circulating platelets. Additional functional analy- ses were performed with platelets, invariably from patients in the major treatment classes.
For 36 of the patients, blood samples could be obtained before and one hour after platelet transfusion. As expected, platelet count increased after transfusion (Online Supplementary Table S4). The clinical efficacy of transfusion was evaluated from the corrected count increment (CCI: [platelet count increment x body surface area]/[number of transfused platelets x 1011]).20 This was adequate for 96% of the patients, as indicated by a CCI value of >7.5 (median: 14.8, IQR: 11.3-18.0).
Flow cytometric analysis of integrin activation and P-selectin expression demonstrated that at one hour after transfusion, platelet responsiveness was improved for most patients (Online Supplementary Figure S2). Whenever possible, platelets were also isolated from the remainder of the transfusion concentrates. It appeared that the activity of the circulating platelets after transfusion approached that of the platelets of the concentrates when triggered with thrombin or CRP-XL. However, the responsiveness to ADP of the circulating platelets after transfusion was high- er than in concentrates (integrin aIIbβ3 activation, P=0.002). The improved platelet responses after transfusion under- lined the low responsiveness of the autologous platelets after chemotherapy.
Impaired platelet responsiveness during myelosuppression
To determine whether the reduced platelet responsive- ness was linked to the treatment phase, flow cytometric
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